Substituted bicyclic pyrimidine-based compounds and compositions and uses thereof

ABSTRACT

Novel C-2-substituted bicyclic compounds of Formula I have been prepared and found to be useful as inhibitors of by inhibiting geranylgeranylation of proteins. 
     
       
         
         
             
             
         
       
     
     The application is directed to these compounds, to compositions comprising these compounds and to their use, in particular as medicaments to cancer and other conditions treatable by inhibiting human geranylgeranylation pyrophosphate hGGPPS activity.

RELATED APPLICATIONS

This application is a continuation of co-pending InternationalApplication No. PCT/CA2018/050091, filed on Jan. 26, 2018, which claimsthe benefit of priority of U.S. Provisional Patent Application No.62/528,601 filed Jul. 5, 2017, and U.S. Provisional Patent ApplicationNo. 62/450,736 filed Jan. 26, 2017 the contents of each of which areincorporated herein by reference in their entirety.

FIELD

The present application relates to novel bicyclic heterocycliccompounds, to processes for their preparation, to compositionscomprising them, and to their use, for example in therapy. Moreparticularly, the application relates to compounds useful in thetreatment of diseases, disorders or conditions that are mediated by highlevels of the isoprenoid metabolites, particularly, the metabolitegeranylgeranyl pyrophosphate (GGPPS).

BACKGROUND

Post-translational modifications of small GTPases with farnesylpyrophosphate (FPP; C15 isoprenoid) or geranylgeranylation pyrophosphate(GGPP; C20 isoprenoid) is commonly known as protein prenylation.Prenylation provides GTPases with the ability to associate specificallywith cellular membranes which have a high concentration of signallingbiomolecules and consequently, participate in a plethora of cellularfunctions, including cell signalings proliferation, and synapticplasticity (FIG. 1a,b ); for a recent review refer to Wang, M.; Casey,P. J. Nature Rev./Mol. Cell Biol. 2016, 77, 110-122.

In the past, inhibition of protein prenylation focused mainly onblocking RAS activation, especially mutated K-RAS, which is a commondriver of oncogenesis, by inhibiting the transferase enzyme FTase thatcatalyses the attachment of FPP to the GTPases, including K-Ras, H-RASand N-Ras proteins. However, a biochemical redundancy mechanism allowsK-Ras activation by GGPP prenylation, which is catalyzed by thetransferase enzyme GGTPase I; consequently, GGTPase I takes over thetask of Ras prenylation, when FTase is inhibited (FIG. 1; Rowinsky, E.K. J. Clin. Oncol. 2006, 24, 2981-2984). It was then suspected thatthere are several mechanisms leading to the escape of FTase inhibition(in addition to the alternative prenylation by GGTPase I), consequentlyattention was re-directed to targeting GGTase I. For example, a geneticsstudy showed that conditional deletion of the gene encoding a β-subunitof GGTase I in myeloid and lung cells, almost completely eliminated theproliferation and tumour formation that accompanies induction of K-Rasexpression, leading to markedly improved survival of mice (Sjogren,A.-K. M. et al. J. Clin. Invest. 2007, 117, 1294-1304). This studysuggested that inhibition of GGPP prenylation may be a useful strategyto treat K-Ras-induced malignancies, in addition to other human diseasesthat are driven by GGPP prenylation of small GTP binding proteins(commonly referred to as GTPases), such as RhoA, RhoB, RhoC, Rac1,cdc-42, R-Ras and Rap1A (Kho, Y. et al. Proc. Natl. Acad Sci. USA 2004,101, 12479-12484).

Current biochemical evidence from Applicant's own research (Pelleieux,S. et al. Isoprenoids and tau pathology in sporadic Alzheimer's disease.Neurobiology of Aging 2018, in press) and other researcher groups[examples include (a) Eckert, G. P. et al. Neurobiol. Disease 2009, 35,252; (b) Hooff, G. P. et al. Biochim. Biophys. Acta 2010, 1801, 896;]also suggests that high intracellular levels of isoprenoids in the brainof Alzheimer's patients is potentially involved in the accumulation ofphosphorylated tau (P-Tau) protein and neuronal damage (FIG. 1b ). P-Tauis the hallmark of neurofibrillary tangle formation in the brain andstrongly implicated in the progression of Alzheimer's disease (He, Z. etal. Nature Medicine 2018, 24, 29-38).

The prenylation cascade from FPP→GGPP→RhoA-cdc42→GSK3-β→phospho-Tau(FIG. 1b ) has been proposed as largely responsible forAlzheimer's-associated tau phosphorylation and tangle formation ofneurons. Therefore, inhibitors of hGGPPS may be valuable therapeuticsfor arresting the progression of Alzheimer's disease or delaying itsonset in pre-symptomatic subjects.

The human enzymes fanesyl pyrophosphate synthase (hFPPS) and humangeranylgeranyl pyrophosphate synthase (hGGPPS) control strategic stepsin the mevalonate pathway (FIGS. 1a and 1b ) and are functionally verysimilar in that, during their catalytic cycle, they both bind apyrophosphate-based substrate (DMAPP, GPP or FPP) and extend itshydrocarbon side chain via the addition of an IPP unit (FIG. 1a ).Consequently, non-selective inhibition of both enzymes by compounds thatare pyrophosphate mimics (e.g. bioisosteres) has been observed. In thepast, drug discovery efforts was based on the presumption thatselectivity in inhibiting hGGPPS over hFPPS is of little therapeuticvalue, since inhibition of hFPPS will inevitably lead to intracellulardepletion of the required substrate for hGGPPS, thus indirectlydecreasing the intracellular levels of the GGPP metabolite (FIG. 1).

Numerous nitrogen-containing bisphosphonate (A-BP) compounds thatselectively inhibit hFPPS have been reported in the literature,including zoledronic acid (ZOL), a clinically useful drug for thetreatment of bone diseases, such as osteoporosis and lytic bone diseasesdue to cancer (Melton, L. J., 3rd et al. J Bone Miner Res 2005, 20,487-493). Interestingly, there is an on-going debate in the scientificand medical communities as to whether (or not) A-BP drugs thatselectively inhibit hFPPS, such as ZOL, are bona fide antitumor agents.Clinical trials with breast cancer (Coleman, R. E. et al. New Engl. J.Med. 365, 1396-1405) and multiple myeloma (MM) patients [(a) Morgan, G.J. et al. Lancet 376, 1989-1999; (b) Morgan, G. J. et al. Blood 119,5374-5383] treated with standard chemotherapy plus ZOL indicatedimproved disease-free survival, although the effects were limited.Additionally, statins that block the mevalonate pathway at the initialHMG-CoA level (FIG. 1b ) were reported to reduce mortality in MMpatients [(a) Mullen, P. J. et al. Nature Rev/Cancer 2016, 16, 716-731.(b) Clendening, J. W. et. A.l Proc Natl Acad Sci USA 2010, 107,15051-15056], ZOL is expected to directly downregulate farnesylation andthat could be one of the mechanisms of action responsible for theantitumor effects, in addition to indirectly downregulatinggeranylgeranylation via depletion of the intracellular levels of FPP(FIG. 1a ).

The geranylgeranylated GTPase proteins (e.g. Rap1A, Rho Ram, Rac andCdc42) play an indispensable role in signal transduction cascades ofcell growth, differentiation, and survival. Inhibition of hGGPPS leadsto the pleiotropic biochemical consequences. For example, decrease inRho kinase responses (as a consequence of statin treatment) has beenimplicated in increased production of endothelium-derived nitric oxide(NO) (Rikitake and Liao Circ Res 2005, 97, 1232-1235). Endothelialdysfunction is characterized as the decreased synthesis, release, and/oractivity of endothelial-derived NO and is believed to be a strongpredictor of cardiovascular disease. Therefore, the regulation of NO byRho may be an important mechanism underlying the cardiovascularprotective effect of statins. Geranylgeranylated GTPases are alsoimplicated in oncogenesis (Sorrentino et al. Nature Cell Biol. 2014, 16,357-366). Inhibition of hGGPPS decreases the migration/metastasis ofhighly invasive breast cancer cells (Dudakovic et al. Invest New Drugs2011, 29, 912-920), induces autophagy in prostate cancer (Wasko et al. JPharamacol Exp Ther 2011, 337, 540-546) and plays a role in the survivalof glioma cells (Yu et al. BMC Cancer 2014, 14:248; doi:10.1186/1471-2407-14-248).

Only a handful of exploratory compounds that are reasonably potentinhibitors of hGGPPS have been reported in the literature. However,based on the reported biological activity and the chemical structures ofthese compounds, none are expected to exhibit the requiredbiopharmaceutical properties for a clinically useful therapeutic agent.Bisphosphonates with long side chains, such as analog A, have been shownto inhibit hGGPPS [(a) Zhang, Y. et al. J. Am. Chem. Soc. 2009, 131,5153-5162. (b) Zhang, Y. et al. Angew. Chem. Int. Ed. 2010, 49,1136-1138]. Substituted biphenyl bisphosphonates such as compound B,have also been described as selective hGGPPS inhibitors, however thiscompound is not very potent (Guo, R. T. et al. Proc Natl Acad Sci USA2007, 104, 10022-10027). Structural mimics of isoprenoids, such as thebranched bisphosphonate digeranyl analog C and the triazole FPP mimic Dare amongst the most potent hGGPPS inhibitors reported to date [(a)Shull, L. W. et al. Bioorg Med Chem 2006, 14, 4130-4136. (b) Barney, R.J. et al. Bioorg Med Chem 2010, 18, 7212-7220. (c) Wills, V. S. et al.ACS Med. Chem. Lett. 2015, 6, 1195-1198]. However, compoundscharacterized by long hydrocarbon chains (such as compound A, C and D),in addition to multiple non-aromatic double bonds (such as compounds Cand D) are highly susceptible to cytochrome P 450 metabolic oxidation(i.e. low metabolic stability) and may also suffer from poor chemicalstability.

PCT patent application publication no. WO 2016/081281 discloseslipophilic bisphosphonate compounds that are reported to inhibit FPPSand/or GGPPS. US patent application US2015/0322099A1 and PCT applicationWO 2014/008407 disclose GGPPS selective inhibitors consisting ofbisphosphonate compounds with one aromatic chain and one aliphaticisoprenoid chain attached via an ether linkage. PCT patent applicationpublication no. WO 2014/176546 teaches that GGPPS inhibitors may beuseful for treating fibrosis, such as pulmonary fibrosis.

The design and synthesis of novel C-6-substituted thienopyrimidine-basedbisphosphonates (ThP-BPs) inhibitors of hFPPS was recently reported.[(a) Leung, C. Y. et al. J. Med. Chem. 2013, 56, 7939-7950. (b) DeSchutter, J. W. et al. J. Med. Chem 2014, 57, 5764-5776.]

SUMMARY

A novel class of bicyclic heterocyclic compounds of Formula I have beenprepared and found to be useful for inhibiting the biosynthesis of GGPPand geranylgeranylation of GTPases, for example via their activity aspotent inhibitors of hGGPPS, which are also moderately selective againsthFPPS.

Potent inhibitors of hGGPPS induce a pronounced effect in blockingcancer cell proliferation, particularly by blocking the proliferation ofmultiple myeloma (MM), chromic myelogenous leukemia cells and othertypes of cancer cell lines and leading to their apoptosis. Theseinhibitors perform far better than potent inhibitors of hFPPS, includingthe commercial drug zoledronic acid (ZOL), and C-6 substitutedthienopyrimidine-based bisphosphonates. The latter are potent inhibitorsof hFPPS, but not hGGPPS, with very similar physicochemical propertiesto the hGGPPS inhibitors disclosed herein.

Inhibitors of hGGPPS may also have a more pronounced effect indownregulating the levels of phosphorylate tau protein (P-Tau) in humanneurons than hFPPS inhibitors. Therefore, inhibitors of hGGPPS may alsobe valuable therapeutic agents for arresting the initiation orprogression of P-Tau-dependent formation of neurofibrillary tangles,which can cause neurodegeneration and are one of the currently knownhallmarks of Alzheimer's disease.

Accordingly, one aspect of the present application includes a compoundof Formula I, or a pharmaceutically acceptable salt, solvate and/orprodrug thereof:

wherein:R is selected from H, C₁₋₂alkyl and C₁₋₂fluoroalkyl;R¹ is a pyrophosphate bioisostere;X is selected from O, CH₂, NH and N(C₁₋₄alkyl);Z and Y are independently selected from S, O, NR³ and CR³R^(3′);Cy¹ is selected from C₆₋₁₀aryl, C₅₋₁₀heteroaryl, C₃₋₁₀cycloalkyl andC₃₋₁₀heterocycloalkyl each of which are unsubstituted or substitutedwith one or two substituents independently selected from halo, cyano,hydroxyl, NH₂, NHC₁₋₆alkyl, NHC₃₋₄cycloalkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl),C₁₋₆fluoroalkyl, C₁₋₆alkyl, C₃₋₆cycloalkyl, C₁₋₆fluoroalkoxy, C₁₋₆alkoxyand C₃₋₆cycloalkoxy;Cy² is selected from C₃₋₁₀cycloalkyl, C₃₋₁₀heterocycloalkyl, C₆₋₁₀aryland C₅₋₁₀heteroaryl, each of which is unsubstituted or substituted withone to three substituents independently selected from halo, cyano,hydroxyl, NH₂, NHC₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), NHC₃₋₆cycloalkyl,C₁₋₆fluoroalkyl, C₁₋₆alkyl, C₃₋₆cycloalkyl, C₁₋₆fluoroalkoxy,C₃₋₆cycloalkoxy, phenyl, C₃₋₆heterocycloalkyl, C₅₋₆heteroaryl andC₁₋₆alkoxy;L is selected from a direct bond, C(O), O, AC(O)(CR⁴R^(4′))_(m)(A′)_(p),ASO₂(CR⁴R^(4′))_(m)(A′)_(p), C(O)A(CR⁴R^(4′))_(m)(A′)_(p) andSO₂A(CR⁴R^(4′))_(m)(A′)_(p);R³ and R^(3′) are independently selected from H, C₃₋₆cycloalkyl andC₁₋₄alkyl, or when the atom to which R³ is attached is sp₂ hybridized,R³ is not present;R⁴ and R^(4′) are independently selected from H, halo, C₁₋₄fluoroalkyl,C₁₋₄alkyl, C₃₋₆cycloalkyl, C₁₋₄fluoroalkoxy, C₃₋₆cycloalkoxy andC₁₋₄alkoxy;m is selected from 0, 1 and 2;p is selected from 0 and 1;A is selected from NH and N(C₁₋₄alkyl);A′ is selected from O, NH and N(C₁₋₄ alkyl) when m is 1 or 2 and A′ isselected from NH and N(C₁₋₄ alkyl) when m is 0; and

represents a single or double bond, provided that two double bonds arenot adjacent to each other.

The present application also includes a compound of Formula I, or apharmaceutically acceptable salt, solvate and/or prodrug thereof:

wherein:R is selected from H, C₁₋₂alkyl and C₁₋₂fluoroalkyl;R¹ is a pyrophosphate bioisostere;X is selected from O, CH₂, NH and N(C₁₋₄alkyl);Z and Y are independently selected from S, O, NR³ and CR³R^(3′);Cy¹ is selected from C₆₋₁₀aryl, C₅₋₁₀heteroaryl, C₃₋₁₀cycloalkyl andC₃₋₁₀heterocycloalkyl, each of which are unsubstituted or substitutedwith one or two substituents independently selected from halo, cyano,hydroxyl, NH₂, NHC₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), C₁₋₆fluoroalkyl,C₁₋₆alkyl, C₁₋₆fluoroalkoxy and C₁₋₆alkoxy;Cy² is selected from C₃₋₁₀cycloalkyl, C₃₋₁₀heterocycloalkyl, C₆₋₁₀aryland C₅₋₁₀heteroaryl, each of which is unsubstituted or substituted withone to three substituents independently selected from halo, cyano,hydroxyl, NH₂, NHC₄alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), C₁₋₆fluoroalkyl,C₁₋₆alkyl, C₁₋₆fluoroalkoxy and C₁₋₆alkoxy;L is selected from a direct bond, C(O), O, AC(O)(CR⁴R^(4′))_(m)(A′)_(p),ASO₂(CR⁴R^(4′))_(m)(A′)_(p), C(O)A(CR⁴R^(4′))_(m)(A′)_(p) andSO₂A(CR⁴R^(4′))_(m)(A′)_(p);R³ and R^(3′) are independently selected from H and C₁₋₄alkyl, or whenthe atom to which R³ is attached is sp₂ hybridized, R³ is not present;R⁴ and R^(4′) are independently selected from H, halo, C₁₋₄fluoroalkyl,C₁₋₄alkyl, C₁₋₄fluoroalkoxy and C₁₋₄alkoxy;m is selected from 0, 1 and 2;p is selected from 0 and 1;A is selected from NH and N(C₁₋₄ alkyl);A′ is selected from O, NH and N(C₁₋₄ alkyl) when m is 1 or 2 and A′ isselected from NH and N(C₁₋₄alkyl) when m is 0; and

represents a single or double bond, provided that two double bonds arenot adjacent to each other.

The present application also includes a composition comprising one ormore compounds of the application and a carrier. In an embodiment, thecomposition is a pharmaceutical composition comprising one or morecompounds of the application and a pharmaceutically acceptable carrier.

The compounds of the application inhibit hGGPPS function. Therefore, thecompounds of the application are useful for treating diseases, disordersor conditions mediated by hGGPPS. Accordingly, the present applicationalso includes a method of treating a disease, disorder or conditionmediated by or through hGGPPS comprising administering a therapeuticallyeffective amount of one or more compounds of the application to asubject in need thereof.

In a further embodiment, the compounds of the application are used asmedicaments. Accordingly, the application also includes a compound ofthe application for use as a medicament.

The present application also includes a method of treating a disease,disorder or condition that is mediated by hGGPPS or is treatable byinhibiting geranylgeranylation comprising administering atherapeutically effective amount of one or more compounds of theapplication to a subject in need thereof. The present application alsoincludes a use of one or more compounds of the application for treatmentof a disease, disorder or condition mediated by hGGPPS or treatable byinhibiting geranylgeranylation as well as a use of one or more compoundsof the application for the preparation of a medicament for treatment ofa disease, disorder or condition mediated by hGGPPS or treatable byinhibiting geranylgeranylation. The application further includes one ormore compounds of the application for use in treating a disease,disorder or condition mediated by hGGPPS or treatable by inhibitinggeranylgeranylation.

The present application also provides evidence that inhibitors of hGGPPSexhibit much stronger antimyeloma effects than inhibitors of hFPPS withequivalent potency and very similar structural and physicochemicalproperties. The mRNA levels of hFPPS and hGGPPS were analyzed in variousMM cell lines reported in the Cancer Cell Line Encyclopedia [refer to:(a) The Cancer Cell Line Encyclopedia and Genomics of Drug Sensitivityin Cancer Investigators. Pharmacogenomic agreement between two cancercell line data sets Nature 2015, 528, 84-87; (b) Barretina, J. et al.The Cancer Cell Line Encyclopedia enables predictive modelling ofanticancer drug sensitivity. Nature 2012, 483, 603-607]. Interestingly,it was found that the mRNA levels of hFPPS were consistentlysignificantly higher than those of hGGPPS in all MM human cells (FIG. 2a). Additionally, the mRNA levels of hFPPS and hGGPPS were analyzed inprimary MM cells taken from bone marrow specimens of MM patientparticipating in a large clinical trial (data from CD138-selected MMcells from 724 patient bone marrow specimens obtained at diagnosis;CoMMpass IA10 Clinical trial data). It was confirmed that mRNAexpression of hFPPS is also significantly higher than of hGGPPS intumors from these patients. (FIG. 2b ). Collectively, these findings inMM cells and myeloma patients suggest a much higher (%) targetengagement of the human GGPPS in vivo and consequently, a more effectiveinhibition of GGPP-dependent activation of GTPases in vivo upontreatment with an hGGPPS inhibitor than with an equipotent andphysicochemically equivalent inhibitor of hFPPS.

The present application also provides evidence that the compoundsdisclosed herein, which are inhibitors of hGGPPS, also block cancer cellproliferation of many types of cancers (FIG. 3c ), including cancer celllines that are resistant to current chemotherapeutic drugs, for example,doxorubicin, such as the multidrug resistant ovarian cancer cells(ADR-RES; FIG. 3b ). However, the compounds disclosed herein, which areinhibitors of hGGPPS, are significantly less toxic to normal humanbronchial cells (NHBE) than the broad-spectrum antitumor agentdoxorubicin (FIG. 3a ).

The present application also provides evidence that the compoundsdisclosed herein, which are inhibitors of hGGPPS, also blockgeranylgeranylation of Rap 1A in MM cancer cells (FIG. 8a ), as well asthe peripheral blood of Vk*MYC transgenic mice (FIG. 8b ), withoutcausing overt toxicity. These mice are an ideal MM disease model, shownto clinically recapitulate the human MM disease.

The present application also provides evidence that the compoundsdisclosed herein, which are inhibitors of hGGPPS, are metabolicallyhighly stable in male CD-1 mouse liver microsomes (MLM), Sprague-Dawleyrat liver microsomes (RLM) and human liver microsomes (HLM).

In an embodiment, the disease, disorder or condition mediated by hGGPPSor treatable by inhibiting geranylgeranylation is a neoplastic disorder.In an embodiment, the treatment is in an amount effective to ameliorateat least one symptom of the neoplastic disorder, for example, reducedcell proliferation or reduced tumor mass, volume or distribution in asubject in need of such treatment.

Bisphosphonate inhibitors of hGGPPS are also known to block activity ofgeranylgeranylated proteins, such as cdc42, Rac, and Rho in osteoclasts,which is directly related to the antiresportive effects in bone.Therefore, the compounds disclosed herein, which are bisphosphonateinhibitors of hGGPPS that bind with equivalent affinity to bone aszoledronic acid and riserdonic acid, as determined using the ¹HNMR-based method published by Novartis (see (a) Jahnke, W. et al.ChemMedChem 2010, 5, 770-776 and (b) Angew. Chem. Int. Ed. 2015, 54,14575-14579) may be used for the treatment of bone disorders such asosteoporosis and cancer-related lytic bone disease. Therefore, in anembodiment, the disease, disorder or condition mediated by hGGPPS, ortreatable by inhibiting geranylgeranylation, is a bone disorder, such asosteoporosis and cancer-related lytic bone disease.

In an embodiment, the disease, disorder or condition mediated by hGGPPS,or treatable by inhibiting geranylgeranylation, is cancer.

In an embodiment, the disease, disorder or condition mediated by hGGPPS,or treatable by inhibiting geranylgeranylation, is a disease, disorderor condition associated with an uncontrolled and/or abnormal cellularactivity affected directly or indirectly by an inhibition of hGGPPS. Inanother embodiment, the uncontrolled and/or abnormal cellular activitythat is affected directly or indirectly by inhibition of hGGPPS isproliferative activity in a cell.

The application also includes a method of inhibiting proliferativeactivity in a cell, comprising administering or delivering an effectiveamount of one or more compounds of the application to the cell.

In an embodiment, the disease, disorder or condition mediated by hGGPPS,or treatable by inhibiting geranylgeranylation, are taupathies, leadingto neurodegeneration. Taupathies have been strongly implicated with theonset and progression of Alzheimer's Disease. Therefore, in anembodiment, the disease, disorder or condition mediated by hGGPPS, ortreatable by inhibiting geranylgeranylation, is Alzheimer's Disease.

In a further embodiment the disease, disorder or condition mediated byhGGPPS, or treatable by inhibiting geranylgeranylation, is cancer andthe one or more compounds of the application are administered incombination with one or more additional cancer treatments. In anotherembodiment, the additional cancer treatment is selected fromradiotherapy, chemotherapy, targeted therapies, such as antibodytherapies, and small molecule therapies, such as tyrosine-kinaseinhibitors, immunotherapy, hormonal therapy and anti-angiogenictherapies.

In other embodiments, the present compounds are useful medically acrossthe broad range of medical conditions that are connected with thenumerous pathways regulated in full or in part by the prenylation ofproteins including, cancer, inflammation and cardiovascular diseases.

In other embodiments, the present compound may be most useful for thetreatment of cancer, and in particular in treating both the primarymalignancy of multiple myeloma, as well as its characteristic lytic bonedisease.

The application additionally provides a process for the preparation ofcompounds of the application. General and specific processes arediscussed in more detail below and set forth in the Examples below.

Other features and advantages of the present application will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating embodiments of the application, are given byway of illustration only and the scope of the claims should not belimited by these embodiments, but should be given the broadestinterpretation consistent with the description as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

The application will be described in greater detail with reference tothe drawing in which:

FIG. 1 contains schematics showing (a) the biosynthesis of isoprenoids;the structures of metabolites associated with isoprenois, including FPPand GGPP are indicated, and (b) the mevalonate pathways; current classesof drugs that modulate the function of key enzymes (i.e. the statins andthe bisphosphonates) are indicated as well as the enzymes hFPPS andhGGPS.

FIG. 2 shows data from (a) the mRNA levels of hFPPS and hGGPPS invarious MM cell lines reported in the Cancer Cell Line Encyclopedia and(b) the CoMM IA10 clinical trial, which shows the mRNA expression levelof hFPPS is higher than that of hGGPPS in bone marrow samples ofmultiple myeloma patients.

FIG. 3a,b shows a comparison of the antiproliferative effect ofdoxorubicin and two exemplary compounds I-37 and I-39 on (a) normalhuman bronchial cells (NHBE cells), (b) ovarian cancer cells expressinghigh levels of multi-drug resistance pumps (ADR-RES cells). FIG. 3cshows the antiproliferative effect exemplary compounds I-37 in variouscancer cell lines; the cell lines used are described in Table 4.

FIG. 4 shows the antitumour effects of exemplary compound I-6 andzolendronate in chronic myelogenous leukemia cells (K562) and acutemonocytic leukemia cells (MOLM-13).

FIG. 5 shows the antitumour effects of exemplary compounds I-6, I-34,I-7, I-35, I-36, I-39, I-37, and I-40 in multiple myeloma cells(RPMI-8226).

FIG. 6 shows an example of flow cytometry data for detection of cellapoptosis, using the multiple myeloma cell line RPMI-8226. Controlexperiments with untreated multiple myeloma cells and cells treated withthe drug Vercade (a potent antitumor agent for the treatment of multiplemyeloma) were run in parallel.

FIG. 7 contains inhibitory activity (IC₅₀ values) of prior art compound6-I (an inhibitor of hFPPS; of WO2014/078957) compared to the exemplaryhGGPPS inhibitor compound I-5 of the present application, as well astheir physicochemical properties including their Clog P values and C-18reverse phase HPLC retention times; the latter properties are reflectiveof the physicochemical similarities between the two compounds I-5 and6-I

FIG. 8 contains the Wester blot data indication a dose dependentinhibition of Rap 1A prenylation upon treatment with inhibitor compoundI-37, more specifically, (a) shows the intracellular levels ofunprenylated Rap1A in multiple myeloma cells RPMI-8226 treated withinhibitor compound I-37 over a 48 hour period and (b) the intracellularlevels of unprenylated Rap 1A in the peripheral blood of Vk*MYCtransgenic mice, with advanced MM disease symptoms after treatment withinhibitor compound I-37 at 1 mg/Kg and 5 mg/Kg dose with 17 doses over aperiod or 16 days (with a drug holiday during the weekends within thatperiod).

DETAILED DESCRIPTION I. Definitions

Unless otherwise indicated, the definitions and embodiments described inthis and other sections are intended to be applicable to all embodimentsand aspects of the application herein described for which they aresuitable as would be understood by a person skilled in the art. Unlessotherwise specified within this application or unless a person skilledin the art would understand otherwise, the nomenclature used in thisapplication generally follows the examples and rules stated in“Nomenclature of Organic Chemistry” (Pergamon Press, 1979), Sections A,B, C, D, E, F, and H. Optionally, a name of a compound may be generatedusing a chemical naming program such as ChemDraw, ACD/ChemSketch,Version 5.09/September 2001, Advanced Chemistry Development, Inc.,Toronto, Canada.

The term “compound of the application” or “compound of the presentapplication” and the like as used herein refers to a compound of FormulaI or pharmaceutically acceptable salts, solvates and/or radiolabeledversions thereof.

The term “composition of the application” or “composition of the presentapplication” and the like as used herein refers to a composition, suchas a pharmaceutical composition, comprising one or more compounds ofFormula I, or pharmaceutically acceptable salts, solvates and/orradiolabeled versions thereof.

The term “and/or” as used herein means that the listed items arepresent, or used, individually or in combination. In effect, this termmeans that “at least one of” or “one or more” of the listed items isused or present. The term “and/or” with respect to pharmaceuticallyacceptable salts, solvates and/or radiolabeled versions thereof meansthat the compounds of the application exist as individual salts,hydrates or radiolabeled versions, as well as a combination of, forexample, a salt of a solvate of a compound of the application or a saltof a radiolabeled version of a compound of the application.

As used in the present application, the singular forms “a”, “an” and“the” include plural references unless the content clearly dictatesotherwise. For example, an embodiment including “a compound” should beunderstood to present certain aspects with one compound, or two or moreadditional compounds.

In embodiments comprising an “additional” or “second” component, such asan additional or second compound, the second component as used herein ischemically different from the other components or first component. A“third” component is different from the other, first, and secondcomponents, and further enumerated or “additional” components aresimilarly different.

In understanding the scope of the present application, the term“comprising” (and any form of comprising, such as “comprise” and“comprises”), “having” (and any form of having, such as “have” and“has”), “including” (and any form of including, such as “include” and“includes”) or “containing” (and any form of containing, such as“contain” and “contains”), are inclusive or open-ended and do notexclude additional, unrecited elements, or process steps.

The term “consisting” and its derivatives as used herein are intended tobe closed terms that specify the presence of the stated features,elements, components, groups, integers, and/or steps, and also excludethe presence of other unstated features, elements, components, groups,integers and/or steps.

The term “consisting essentially of” as used herein is intended tospecify the presence of the stated features, elements, components,groups, integers, and/or steps as well as those that do not materiallyaffect the basic and novel characteristic(s) of features, elements,components, groups, integers, and/or steps.

The term “suitable” as used herein means that the selection of theparticular compound or conditions would depend on the specific syntheticmanipulation to be performed, the identity of the molecule(s) to betransformed and/or the specific use for the compound, but the selectionwould be well within the skill of a person trained in the art.

In embodiments of the present application, the compounds describedherein may have at least one asymmetric center. Where compounds possessmore than one asymmetric center, they may exist as diastereomers. It isto be understood that all such isomers and mixtures thereof in anyproportion are encompassed within the scope of the present application.It is to be further understood that while the stereochemistry of thecompounds may be as shown in any given compound named or depictedherein, such compounds may also contain certain amounts (for example,less than 20%, suitably less than 10%, more suitably less than 5%) ofcompounds of the present application having an alternatestereochemistry. It is intended that any optical isomers, as separated,pure or partially purified optical isomers or racemic mixtures thereofare included within the scope of the present application.

The compounds of the present application may also exist in differenttautomeric forms and it is intended that any tautomers which thecompounds form, as well as mixtures thereof, are included within thescope of the present application.

The compounds of the present application may further exist in varyingpolymorphic forms and it is contemplated that any polymorphs, ormixtures thereof, which form are included within the scope of thepresent application.

Terms of degree such as “substantially”, “about” and “approximately” asused herein mean a reasonable amount of deviation of the modified termsuch that the end result is not significantly changed. These terms ofdegree should be construed as including a deviation of at least ±5% ofthe modified term if this deviation would not negate the meaning of theword it modifies or unless the context suggests otherwise to a personskilled in the art.

The expression “proceed to a sufficient extent” as used herein withreference to the reactions or process steps disclosed herein means thatthe reactions or process steps proceed to an extent that conversion ofthe starting material or substrate to product is sufficient for thegiven reaction. Conversion may be sufficient when greater than about 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95or 100% of the starting material or substrate is converted to product.

Based on IUPAC rules, numbering of the atoms around the biclyclicheterocyclic core of Formula I will vary depending on the exactstructure. For simplicity, references to C-2 and C-4 carbons areassigned based on the pyrimidine ring only as shown below:

The term “alkyl” as used herein, whether it is used alone or as part ofanother group, means straight or branched chain, saturated alkyl groups.The number of carbon atoms that are possible in the referenced alkylgroup are indicated by the prefix “C_(n1-n2)”. For example, the termC₁₋₁₀alkyl means an alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10carbon atoms.

The term “alkylene”, whether it is used alone or as part of anothergroup, means straight or branched chain, saturated alkylene group, thatis, a saturated carbon chain that contains substituents on two of itsends. The number of carbon atoms that are possible in the referencedalkylene group are indicated by the prefix “C_(n1-n2)”. For example, theterm C₀₋₆alkylene means an alkylene group is not present (“C₀alkylene”)or an alkylene group having 1, 2, 3, 4, 5 or 6 carbon atoms.

The term “alkenyl” as used herein, whether it is used alone or as partof another group, means straight or branched chain, unsaturated alkylgroups containing at least one double bond. The number of carbon atomsthat are possible in the referenced alkylene group are indicated by theprefix “C_(n1-n2)”. For example, the term C₂₋₆alkenyl means an alkenylgroup having 2, 3, 4, 5 or 6 carbon atoms and at least one double bond.

The term “haloalkyl” as used herein refers to an alkyl group wherein oneor more, including all of the hydrogen atoms are replaced by a halogenatom. In an embodiment, the halogen is fluorine, in which case thehaloalkyl is referred to herein as a “fluoroalkyl” group.

The term “alkoxy” as used herein, whether it is used alone or as part ofanother group, refers to the group “alkyl-O—” or “—O-alkyl”. The termC₁₋₁₀alkoxy means an alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10carbon atoms bonded to an oxygen atom. Exemplary alkoxy groups includewithout limitation methoxy, ethoxy, propoxy, isopropoxy, butoxy,t-butoxy and isobutoxy.

The term “cycloalkyl,” as used herein, whether it is used alone or aspart of another group, means a saturated carbocyclic group containing anumber of carbon atoms and one or more rings. The number of carbon atomsthat are possible in the referenced cycloalkyl group are indicated bythe numerical prefix “C_(n1-n2)”. For example, the term C₃₋₁₀cycloalkylmeans a cycloalkyl group having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

The term “cycloalkoxy,” as used herein, whether it is used alone or aspart of another group, refers to the group “cycloalkyl-O—” or“—O-cycloalkyl”. The number of carbon atoms that are possible in thereferenced cycloalkyl group are indicated by the numerical prefix“C_(n1-n2)”. For example, the term C₃₋₁₀cycloalkoxy means an cycloalkylgroup having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms bonded to an oxygenatom.

The term “aryl” as used herein, whether it is used alone or as part ofanother group, refers to carbocyclic groups containing at least onearomatic ring. Aryl groups contain one or more than one ring. The numberof carbon atoms that are possible in the referenced aryl group areindicated by the numerical prefix “C_(n1-n2)”. For example, the termC₆₋₁₀aryl means an aryl group having 6, 7, 8, 9 or 10 carbon atoms.

The term “heterocycloalkyl” as used herein, whether it is used alone oras part of another group, refers to non-aromatic cyclic groupscontaining 3 to 10 atoms, and at least one ring in which one or more ofthe atoms are a heteromoiety selected from O, S, N, NH and NC₁₋₆alkyl.Heterocycloalkyl groups are either saturated or unsaturated (i.e.contain one or more double bonds) and contain one or more than one ring.When a heterocycloalkyl group contains more than one ring, the rings maybe fused, bridged, spirofused or linked by a bond. When aheterocycloalkyl group contains the prefix C_(n1-n2) this prefixindicates the number of carbon atoms in the corresponding carbocyclicgroup, in which one or more of the ring atoms is replaced with aheteromoiety as defined above.

The term “heteroaryl” as used herein refers to cyclic groups containingfrom 5 to 10 atoms, at least one aromatic ring and at least one aheteromoiety selected from O, S, N, NH and NC₁₋₆alkyl. Heteroaryl groupscontain one or more than one ring. When a heteroaryl group contains morethan one ring, the rings may be fused, bridged, spirofused or linked bya bond. When a heteroaryl group contains the prefix C_(n1-n2) thisprefix indicates the number of carbon atoms in the corresponding arylgroup, in which one or more, suitably 1 to 5, of the ring atoms isreplaced with a heteromoiety as defined above.

A 5-membered heteroaryl is a heteroaryl with a ring having five ringatoms, wherein 1, 2 or 3 ring atoms are a heteromoiety selected from O,S, NH and NC₁₋₆alkyl.

A 6-membered heteroaryl is a heteroaryl with a ring having six ringatoms wherein 1, 2 or 3 ring atoms are a heteromoiety selected from O,S, N, NH and NC₁₋₆alkyl.

A 5-membered heterocycloalkyl is a heterocycloalkyl with a ring havingfive ring atoms, wherein 1, 2 or 3 ring atoms are a heteromoietyselected from O, S, NH and NC₁₋₆alkyl.

A 6-membered heterocycloalkyl is a heterocycloalkyl with a ring havingsix ring atoms wherein 1, 2 or 3 ring atoms are a heteromoiety selectedfrom O, S, N, NH and NC₁₋₆alkyl.

All cyclic groups, including aryl and cyclo a groups, contain one ormore than one ring (i.e. are polycyclic). When a cyclic group containsmore than one ring, the rings may be fused, bridged or spirofused.

A first ring being “fused” with a second ring means the first ring andthe second ring share two adjacent atoms there between.

A first ring being “bridged” with a second ring means the first ring andthe second ring share two non-adjacent atoms there between.

A first ring being “spirofused” with a second ring means the first ringand the second ring share one atom there between.

As a prefix, the term “substituted” as used herein refers to astructure, molecule or group in which one or more available hydrogenatoms are replaced with one or more other chemical groups.

As a suffix, the term “substituted” as used herein in relation to afirst structure, molecule or group, followed by one or more variables ornames of chemical groups, refers to a second structure, molecule orgroup that results from replacing one or more available hydrogens of thefirst structure, molecule or group with the one or more variables ornamed chemical groups.

The term “available”, as in “available hydrogen atoms” or “availableatoms” refers to atoms that would be known to a person skilled in theart to be capable of replacement by another atom or substituent.

The term “optionally substituted” refers to groups, structures, ormolecules that are either unsubstituted or are substituted with one ormore substituents.

The term “sp₂ hybridized” as used herein refers to an atom that bondedto one of its neighboring atoms via a double bond.

The term “pyrophosphate bioisostere” as used herein refers to a chemicalsubstituent or group that mimics the pyrophosphate functionality interms of physical and/or chemical properties and which produces broadlysimilar biological properties to the pyrophosphate group.

The term “pyrophosphate” also known as “diphosphate” as used hereinrefers to the chemical group shown below, which is part of the naturalsubstare/metabolite and product of hGGPPS. It will be understood bythose skilled in the art that such a moiety can be ionized, depending onthe pH of its environment, to the mono, di or tri anion

The term “amine” or “amino,” as used herein, whether it is used alone oras part of another group, refers to groups of the general formula NRR′,wherein R and R′ are each independently selected from hydrogen or a analkyl group, such as C₁₋₆alkyl.

The terms “halo” or “halogen” as used herein, whether it is used aloneor as part of another group, refers to a halogen atom and includesfluoro, chloro and bromo.

The term “protecting group” or “PG” and the like as used herein refersto a chemical moiety which protects or masks a reactive portion of amolecule to prevent side reactions in those reactive portions of themolecule, while manipulating or reacting a different portion of themolecule. After the manipulation or reaction is complete, the protectinggroup is removed under conditions that do not degrade or decompose theremaining portions of the molecule. The selection of a suitableprotecting group can be made by a person skilled in the art. Manyconventional protecting groups are known in the art, for example asdescribed in “Protective Groups in Organic Chemistry” McOmie, J. F. W.Ed., Plenum Press, 1973, in “Protective Groups in Organic Synthesis”, T.W. Green, P. G. M. Wuts, Wiley-Interscience, New York, (4^(th) Edition,2007) and in Kocienski, P. Protecting Groups, 3rd Edition, 2003, GeorgThieme Verlag (The Americas).

The term “cell” as used herein refers to a single cell or a plurality ofcells and includes a cell either in a cell culture or in a subject.

The term “subject” as used herein includes all members of the animalkingdom including mammals, and suitably refers to humans.

The term “pharmaceutically acceptable” means compatible with thetreatment of subjects, for example humans.

The term “pharmaceutically acceptable carrier” means a non-toxicsolvent, dispersant, excipient, adjuvant or other material which ismixed with the active ingredient in order to permit the formation of apharmaceutical composition, i.e., a dosage form capable ofadministration to a subject.

The term “pharmaceutically acceptable salt” means either an acidaddition salt or a base addition salt which is suitable for, orcompatible with, the treatment of subjects.

An acid addition salt suitable for, or compatible with, the treatment ofsubjects is any non-toxic organic or inorganic acid addition salt of anybasic compound. Basic compounds that form acid addition salts include,for example, compounds comprising an amine group. Illustrative inorganicacids which form suitable salts include hydrochloric, hydrobromic,sulfuric, nitric and phosphoric acids, as well as acidic metal saltssuch as sodium monohydrogen orthophosphate and potassium hydrogensulfate. Illustrative organic acids which form suitable salts includemono-, di- and tricarboxylic acids. Illustrative of such organic acidsare, for example, acetic, trifluoroacetic, propionic, glycolic, lactic,pyruvic, malonic, succinic, glutaric, funmaric, malic, tartaric, citric,ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic,cinnamic, mandelic, salicylic, 2-phenoxybenzoic, p-toluenesulfonic acidand other sulfonic acids such as methanesulfonic acid, ethanesulfonicacid and 2-hydroxyethanesulfonic acid. In an embodiment, the mono- ordi-acid salts are formed, and such salts exist in either a hydrated,solvated or substantially anhydrous form. In general, acid additionsalts are more soluble in water and various hydrophilic organicsolvents, and generally demonstrate higher melting points in comparisonto their free base forms. The selection criteria for the appropriatesalt will be known to one skilled in the art. Other non-pharmaceuticallyacceptable salts such as but not limited to oxalates may be used, forexample in the isolation of compounds of the application for laboratoryuse, or for subsequent conversion to a pharmaceutically acceptable acidaddition salt.

A base addition salt suitable for, or compatible with, the treatment ofsubjects is any non-toxic organic or inorganic base addition salt of anyacidic compound. Acidic compounds that form a basic addition saltinclude, for example, compounds comprising a carboxylic acid group.Illustrative inorganic bases which form suitable salts include lithium,sodium, potassium, calcium, magnesium or barium hydroxide as well asammonia. Illustrative organic bases which form suitable salts includealiphatic, alicyclic or aromatic organic amines such as isopropylamine,methylamine, trimethylamine, picoline, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine,caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine,glucosamine, methylglucamine, theobromine, purines, piperazine,piperidine, N-ethylpiperidine, polyamine resins, and the like. Exemplaryorganic bases are isopropylamine, diethylamine, ethanolamine,trimethylamine, dicyclohexylamine, choline, and caffeine. [See, forexample, S. M. Berge, et al. “Pharmaceutical Salts,” J. Pharm. Sci.1977, 66, 1-19]. The selection of the appropriate salt may be useful,for example, so that an ester functionality, if any, elsewhere in acompound is not hydrolyzed. The selection criteria for the appropriatesalt will be known to one skilled in the art.

The term “solvate” as used herein means a compound, or a salt of acompound, wherein molecules of a suitable solvent are incorporated inthe crystal lattice. A suitable solvent is physiologically tolerable atthe dosage administered. Examples of suitable solvents are ethanol,water and the like. When water is the solvent, the molecule is referredto as a “hydrate”. The formation of solvates of the compounds of theapplication will vary depending on the compound and the solvate. Ingeneral, solvates are formed by dissolving the compound in theappropriate solvent and isolating the solvate by cooling or using anantisolvent. The solvate is typically dried or azeotroped under ambientconditions. The selection of suitable conditions to form a particularsolvate can be made by a person skilled in the art.

The term “treating” or “treatment” as used herein and as is wellunderstood in the art, means an approach for obtaining beneficial ordesired results, including clinical results. Beneficial or desiredclinical results include, but are not limited to alleviation oramelioration of one or more symptoms or conditions, diminishment ofextent of disease, stabilized (i.e. not worsening) state of disease,preventing spread of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, diminishment of thereoccurrence of disease, and remission (whether partial or total),whether detectable or undetectable. “Treating” and “treatment” can alsomean prolonging survival as compared to expected survival if notreceiving treatment. “Treating” and “treatment” as used herein alsoinclude prophylactic treatment. For example, a subject with early cancercan be treated to prevent progression, or alternatively a subject inremission can be treated with a compound or composition of theapplication to prevent recurrence. Treatment methods compriseadministering to a subject a therapeutically effective amount of one ormore of the compounds of the application and optionally consist of asingle administration, or alternatively comprise a series ofadministrations. For example, the compounds of the application areadministered at least once a week. However, in another embodiment, thecompounds are administered to the subject from about one time per twoweeks, three weeks or one month. In another embodiment, the compoundsare administered about one time per week to about once daily. In anotherembodiment, the compounds are administered 2, 3, 4, 5 or 6 times daily.The length of the treatment period depends on a variety of factors, suchas the severity of the disease, disorder or condition, the age of thesubject, the concentration and/or the activity of the compounds of theapplication, and/or a combination thereof. It will also be appreciatedthat the effective dosage of the compound used for the treatment mayincrease or decrease over the course of a particular treatment regime.Changes in dosage may result and become apparent by standard diagnosticassays known in the art. In some instances, chronic administration isrequired. For example, the compounds are administered to the subject inan amount and for duration sufficient to treat the subject.

“Palliating” a disease, disorder or condition means that the extentand/or undesirable clinical manifestations of a disease, disorder orcondition are lessened and/or time course of the progression is slowedor lengthened, as compared to not treating the disorder.

The term “prevention” or “prophylaxis”, or synonym thereto, as usedherein refers to a reduction in the risk or probability of a patientbecoming afflicted with a disease, disorder or condition or manifestinga symptom associated with a disease, disorder or condition.

The “disease, disorder or condition” as used herein refers to a disease,disorder or condition treatable by hGGPPS enzyme inhibition andparticularly using a hGGPPS enzyme inhibitor, such as a compound of theapplication herein described.

The term “mediated by hGGPPS” or “treatable by hGGPPS inhibition,” asused herein means that the disease, disorder or condition to be treatedis affected by, modulated by and/or has some biological basis, eitherdirect or indirect, that includes hGGPPS activity, in particular,increased hGGPPS activity such as results from hGGPPS geneoverexpression or hGGPPS protein over-accumulation or over-expression ofproteins that are products of or precursors to hGGPPS-mediated geneexpression. In a broader context, “mediated by hGGPPS” can include thelarge number of diseases that are caused by aberrant prenylation, forexample geranylgeranylation, of proteins, as results from aberranthGGPPS activity. As used herein, hGGPPS refers to the protein identifiedas the human geranylgeranyl pyrophosphate synthase enzyme (Park, J. etal. Frontiers in Chemistry 2014, 2, Article 50;doi:10.3389/fchem.2014.00050).

As used herein, the term “effective amount” or “therapeuticallyeffective amount” means an amount of one or more compounds of theapplication that is effective, at dosages and for periods of timenecessary to achieve the desired result. For example, in the context oftreating a disease, disorder or condition mediated by hGGPPS, aneffective amount is an amount that, for example, increases hGGPPSinhibition compared to the inhibition without administration of the oneor more compounds. In an embodiment, effective amounts vary according tofactors such as the disease state, age, sex and/or weight of thesubject. In a further embodiment, the amount of a given compound orcompounds that will correspond to an effective amount will varydepending upon factors, such as the given drug(s) or compound(s), thepharmaceutical formulation, the route of administration, the type ofcondition, disease or disorder, the identity of the subject beingtreated, and the like, but can nevertheless be routinely determined byone skilled in the art.

The term “administered” as used herein means administration of atherapeutically effective amount of one or more compounds orcompositions of the application to a cell, tissue, organ or subject.

The term “neoplastic disorder” as used herein refers to a disease,disorder or condition characterized by cells that have the capacity forautonomous growth or replication, e.g., an abnormal state or conditioncharacterized by proliferative cell growth. The term “neoplasm” as usedherein refers to a mass of tissue resulting from the abnormal growthand/or division of cells in a subject having a neoplastic disorder.Neoplasms can be benign (such as uterine fibroids and melanocytic nevi),potentially malignant (such as carcinoma in situ) or malignant (i.e.cancer). Exemplary neoplastic disorders include the so-called solidtumours and liquid tumours, including but not limited to carcinoma,sarcoma, metastatic disorders (e.g., tumors arising from the prostate),hematopoietic neoplastic disorders, (e.g., leukemias, lymphomas, myelomaand other malignant plasma cell disorders), metastatic tumors and othercancers.

The term “cancer” as used herein refers to cellular-proliferativedisease states.

II. Compounds and Compositions of the Application

Compounds of the present application were prepared and were found toinhibit uncontrolled and/or abnormal cellular activities affecteddirectly or indirectly by hGGPPS. In particular, compounds of thepresent application exhibited activity as hGGPPS inhibitors, and aretherefore useful in therapy, for example for the treatment of neoplasticdisorders such as cancer.

Compounds of the present application may also exhibit activity inreducing the phosphorylation of tau protein in human neurons, and couldpotentially be useful in therapy, for example in decelerating theprogression or the initiation of phospho-tau-dependent neurofibrillarytangles in the brain, which is strongly associated with Alzheimer'sdisease and other tauopathies.

Accordingly, one aspect of the present application includes a compoundof Formula I, or a pharmaceutically acceptable salt, solvate and/orprodrug thereof:

wherein:R is selected from H, CH₂alkyl and C₁₋₂fluoroalkyl;R¹ is a pyrophosphate bioisostere;X is selected from O, CH₂, NH and N(C₁₋₄alkyl);Z and Y are independently selected from S, O, NR³ and CR³R^(3′);Cy¹ is selected from C₆₋₁₀aryl, C₅₋₁₀heteroaryl, C₃₋₁₀cycloalkyl andC₃₋₁₀heterocycloalkyl, each of which are unsubstituted or substitutedwith one or two substituents independently selected from halo, cyano,hydroxyl, NH₂, NHC₁₋₆alkyl, NHC₃₋₆cycloalkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl),C₁₋₆fluoroalkyl, C₁₋₆alkyl, C₃₋₆cycloalkyl, C₁₋₆fluoroalkoxy,C₃₋₆cycloalkoxy and C₁₋₄alkoxy;Cy² is selected from C₃₋₁₀cycloalkyl, C₃₋₁₀heterocycloalkyl, C₆₋₁₀aryland C₅₋₁₀heteroaryl, each of which is unsubstituted or substituted withone to three substituents independently selected from halo, cyano,hydroxyl, NH₂, NHC₁₋₆alkyl, NHC₃₋₆cycloalkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl),C₁₋₆fluoroalkyl, C₁₋₆alkyl, C₃₋₆cycloalkyl, C₁₋₆fluoroalkoxy,C₃₋₆cycloalkoxy, phenyl, C₃₋₆heterocycloalkyl, C₅₋₆heteroaryl andC₁₋₆alkoxy;L is selected from a direct bond, C(O), O, AC(O)(CR⁴R^(4′))_(m)(A′)_(p),ASO₂(CR⁴R^(4′))_(m)(A′)_(p), C(O)A(CR⁴R^(4′))_(m)(A′)_(p) andSO₂A(CR⁴R^(4′))_(m)(A′)_(p);R³ and R^(3′) are independently selected from H, C₃₋₆cycloalkyl andC₁₋₄alkyl, or when the atom to which R³ is attached is sp₂ hybridized,R³ is not present;R⁴ and R^(4′) are independently selected from H, halo, C₁₋₄fluoroalkyl,C₁₋₄alkyl, C₃₋₆cycloalkyl, C₁₋₄fluoroalkoxy, C₃₋₆cycloalkoxy andC₁₋₄alkoxy;m is selected from 0, 1 and 2;p is selected from 0 and 1;A is selected from NH and N(C₁₋₄ alkyl);A′ is selected from O, NH and N(C₁₋₄ alkyl) when m is 1 or 2 and A′ isselected from NH and N(C₁₋₄alkyl) when m is 0; and

represents a single or double bond, provided that two double bonds arenot adjacent to each other.

In some embodiments, R is H, CH₃ or CF₃. In some embodiments R is H.

The group R¹ is any known pyrophosphate bioisostere. In someembodiments, the pyrophosphate bioisostere is selected from one of thefollowing groups:

(a) a bisphosphonate, bisphosphonate ester, or analog or bioisostere ofa bisphosphonate or bisphosphonate ester of the formula(CR⁵R^(5′))PO(OR⁶)₂, wherein R⁵ is a selected from PO(OR^(6′))₂ andphosphate and phosphate ester bioisosteres selected from CO₂R^(6′),C(O)NHR⁷, SO₃R⁷, SO₂NHR⁷ and other such bioisosteres known in the art,for example as described in a review by Elliott, T. S. et al. The use ofphosphate bioisosteres in medicinal chemistry and chemical biology.Chem. Med. Comm. 2012, 3, 735-751; R^(5′) is selected from H, OH andhalo; R⁶ and R^(6′) are independently selected from H and C₁₋₆alkyl andR⁷ is selected from H, OH and C₁₋₆alkyl; and(b) an α,γ-diketo acid (shown below) or a heterocyclic moiety that canserve as a bioisostere of a diketo acid, such as those employed in thedesign of HIV integrase active site inhibitors. Examples include (butare not limited) to compounds described in the EP 1422218, WO2002/30426,WO2002/30930, WO2002/30931 and WO2002/36734.

In some embodiments, R¹ is (CR⁵R^(5′))PO(OR⁶)₂, wherein R⁵ is selectedfrom PO(OR^(6′))₂. CO₂R^(6′), C(O)NHR⁷, SO₃R⁷, SO₂NHR⁷; R^(5′) isselected from H, OH and halo; R⁶ and R^(6′) are independently selectedfrom H and C₁₋₆alkyl; and R⁷ is selected from H, OH and C₁₋₆alkyl. Insome embodiments, R¹ is (CR⁵R^(5′))PO(OR⁶)₂, wherein R⁵ is selected fromPO(OR^(6′))₂, CO₂R^(6′), C(O)NHR⁷, SO₃R⁷, SO₂NHR⁷; R^(5′) is H; R⁶ andR^(6′) are independently selected from H and CH₃; and R⁷ is selectedfrom H, OH and CH₃. In some embodiments, R¹ is (CR⁵R^(5′))PO(OR⁶)₂,wherein R⁵ is PO(OR^(6′))₂, R^(5′) is H; and R⁶ and R^(6′) are both H.

In some embodiments, X is selected from O, CH₂, NH and NCH₃. In someembodiments X is NH.

In some embodiments, the compounds of Formula I have the followingstructure:

wherein Z is selected from S, O, N and CH; Y is selected from S, O, NHand CH₂; and X, R¹, Cy¹, L and Cy² are as defined above.

In some embodiments, the bicyclic core structure in the compounds ofFormula I is selected from any one of the structural isomers shownbelow:

In some embodiments, the compounds of Formula I have the followingstructure:

wherein Y is selected from S, O, NH and CH₂; and X, R¹, Cy¹, L and Cy²are as defined above.

In some embodiments, the compounds of the application arethieno[2,3-d]pyrimidines, i.e. Z is CH and Y is S and the compounds ofFormula I have the following structure:

wherein X, R¹, Cy¹, L and Cy² are as defined above.

In some embodiments, the compounds of the application arethieno[3,2-d]pyrimidin-4-amine, i.e. Z is S and Y is CH and thecompounds of Formula I have the following structure:

wherein X, R¹, Cy¹, L and Cy² are as defined above.

In some embodiments, the compounds of the application arethiazolo[5,4-d]pyrimidin-7-amine, i.e. Z is N and Y is S and thecompounds of Formula I have the following structure:

wherein X, R¹, Cy¹, L and Cy² are as defined above.

In some embodiments, the compounds of the application are9H-purin-6-amine, i.e. Z is N and Y is NR³ and the compounds of FormulaI have the following structure:

wherein X, R¹, R³, Cy¹, L and Cy² are as defined above.

In some embodiments, the compounds of the application are7H-pyrrolo[2,3-d]pyrimidin-4-amine, i.e. Z is CH and Y is NR³ and thecompounds of Formula I have the following structure:

wherein X, R¹, R³, Cy¹, L and Cy² are as defined above.

In some embodiments, Cy¹ is selected from phenyl, C₃₋₆cycloalkyl,C₅₋₁₀heteroaryl and C₅₋₁₀heterocycloalkyl. In some embodiments, Cy¹ isselected from phenyl, naphthyl, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, 5-membered heteroaryl, 6-membered heteroaryl, 10-memberedheteroaryl, 5-membered heterocycloalkyl and 6-membered heterocycloalkyl.In some embodiments, Cy¹ is selected from C₆₋₁₀aryl and C₅₋₁₀heteroaryl.In some embodiments, Cy¹ is selected from phenyl, thienyl, furyl,pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl,isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl,1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl,1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl,1,3,4-oxadiazolyl, pyridinyl, pyrazinyl, pyrimidinyl, triazinyl,pyrrolidinyl, piperazinyl, piperidinyl, morpholinyl and pyridazinyl,each of which is unsubstituted or substituted with one or twosubstituents. In some embodiments, Cy¹ is selected from phenyl, thienyl,furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl,isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl,1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl,1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl,1,3,4-thiadiazolyl, 1,3,4-oxadiazolyl, pyridinyl, pyrazinyl,pyrimidinyl, triazinyl and pyridazinyl, each of which is unsubstitutedor substituted with one or two substituents. In some embodiments, Cy¹ isselected from phenyl, thienyl, furyl, pyrrolyl, pyridinyl, pyrazinyl,pyrrolidinyl, piperazinyl, piperidinyl and pyrimidinyl, each of which isunsubstituted or substituted with one substituent. In some embodiments,Cy¹ is selected from phenyl, thienyl, furyl, pyrrolyl, pyridinyl,pyrazinyl and pyrimidinyl, each of which is unsubstituted or substitutedwith one substituent. In some embodiment Cy¹ is unsubstituted phenyl.

In some embodiments, L is selected from a direct bond, C(O), O,AC(O)(CR⁴R^(4′))_(m)(A′)_(p), ASO₂(CR⁴R^(4′))_(m)(A′)_(p),C(O)A(CR⁴R^(4′))_(m)(A′)_(p) and SO₂A(CR⁴R^(4′))_(m)(A′)_(p). In someembodiments, L is selected from a direct bond,NHC(O)(CR⁴R^(4′))_(m)(A′)_(p), NHSO₂(CR⁴R^(4′))_(m)(A′)_(p),C(O)NH(CR⁴R^(4′))_(m)(A′)_(p) and SO₂NH(CR⁴R^(4′))_(m)(A′)_(p). In someembodiments, L is selected from a direct bond, NHSO₂, SO₂NH, NHC(O),NHC(O)CH₂O, NHC(O)CH(OCH₃), NHC(O)CH(CH₃), NHC(O)CH₂, NHC(O)CH₂CH₂,NHC(O)C(CF₃)(OCH₃)CH₂, C(O)NH, C(O) and NHC(O)NH.

In some embodiments, R³ and R^(3′) are independently selected from H andC₁₋₄alkyl, or when the atom to which R³ is attached is sp₂ hybridized,R³ is not present. In some embodiments, R³ and R^(3′) are independentlyselected from H and CH₃, or when the atom to which R³ is attached is sp₂hybridized, R³ is not present.

In some embodiments, R⁴ and R^(4′) are independently selected from H, F,Cl, CF₃, CH₃, CF₃O and CH₃O. In some embodiments, R⁴ and R^(4′) areindependently selected from H, F, CF₃, CH₃, CF₃O and CH₃O. In someembodiments at least one of R⁴ and R^(4′) is H. In some embodiments bothof R⁴ and R^(4′) are H. In some embodiments neither of R⁴ and R^(4′) areH.

In some embodiments, A is selected from NH and NCH₃. In someembodiments, A is NH.

In some embodiments, A′ is selected from O, NH and NCH₃ when m is 1 or2. In some embodiments, A′ is O or NH when m is 1 or 2. In someembodiments, A′ is selected from NH and NCH₃ when m is 0. In someembodiments, A′ is NH when m is 0.

In some embodiments, m is selected from 0 and 1. In some embodiments mis 0.

In some embodiments, m and p are both 0. In some embodiments, m and pare both 1. In some embodiments, m is 0 and p is 1.

In some embodiments, Cy² is selected from phenyl, C₃₋₆cycloalkyl,C₅₋₁₀heteroaryl and C₅₋₁₀heterocycloalkyl. In some embodiments, Cy² isselected from phenyl, naphthyl, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, 5-membered heteroaryl, 6-membered heteroaryl, 10-memberedheteroaryl, 5-membered heterocycloalkyl and 6-membered heterocycloalkyl.In some embodiments, Cy² is selected from phenyl, naphthyl, cyclopropyl,cyclopentyl, cyclohexyl, thienyl, furyl, pyrrolyl, imidazolyl,thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl,1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl,1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl,1,3,4-thiadiazolyl, 1,3,4-oxadiazolyl, pyridinyl, pyrazinyl,pyrimidinyl, triazinyl, pyridazinyl, piperazinyl, aziridinyl, oxiranyl,thiiranyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrrolinyl,imidazolidinyl, pyrazolidinyl, pyrazolinyl, dioxolanyl, sulfolanyl,2,3-dihydrofuranyl, 2,5-dihydrofuranyl, tetrahydrofuranyl, thiophanyl,piperidinyl, 1,2,3,6-tetrahydro-pyridinyl, piperazinyl, morpholinyl,thiomorpholinyl, pyranyl, thiopyranyl, 2,3-dihydropyranyl,tetrahydropyranyl, 1,4-dihydropyridinyl, 1,4-dioxanyl, 1,3-dioxanyl,dioxanyl, homopiperidinyl, 2,3,4,7-tetrahydro-1H-azepinyl,homopiperazinyl, 1,3-dioxepanyl, 4,7-dihydro-1,3-dioxepinyl,hexamethylene oxide, naphthyl and quinolinyl. In some embodiments, Cy²is selected from phenyl, naphthyl, cyclohexyl, cyclopropyl, thienyl,piperidinyl, furyl, pyrrolyl, pyridinyl, pyrazinyl, pyrimidinyl,morpholinyl and quinolinyl. In some embodiments, Cy² is selected fromphenyl, naphthyl, cyclohexyl, cyclopropyl, thienyl, piperidinyl, furyl,pyrrolyl, pyridinyl, pyrazinyl, pyrimidinyl and quinolinyl. In someembodiments, Cy² is selected from phenyl, thienyl, pyridinyl,piperidinyl, cyclopropyl, quinolinyl, morpholinyl and cyclohexyl. Insome embodiments, Cy² is selected from phenyl, thienyl, pyridinyl,piperidinyl, cyclopropyl, quinolinyl and cyclohexyl.

In some embodiments, Cy¹ is unsubstituted or substituted one substituentselected from Cl, F, NHCH₃, N(CH₃)₂, CF₃, CH₃, CH₃CH₂, (CH₃)₂CH₂, CH₃O,CH₃CH₂O, (CH₃)₂CH₂O, CF₃O and CF₃O. In some embodiments Cy¹ isunsubstituted.

In some embodiments, Cy² is unsubstituted or substituted with 1-3substituents independently selected from Cl, F, phenyl, cyano, hydroxyl,NH₂, NHCH₃, N(CH₃)₂, C₁₋₄fluoroalkyl, C₁₋₄alkyl, C₁₋₄fluoroalkoxy andC₁₋₄alkoxy. In some embodiments, Cy² is unsubstituted or substitutedwith 1-2 substituents independently selected from phenyl, Cl, F, NHCH₃,N(CH₃)₂, C₁₋₃fluoroalkyl, C₁₋₃alkyl, C₁₋₃fluoroalkoxy and C₁₋₃alkoxy. Insome embodiments, Cy² is unsubstituted or substituted with 1-2substituents independently selected from Cl, F, NHCH₃, N(CH₃)₂,C₁₋₃fluoroalkyl, C₁₋₃alkyl, C₁₋₂fluoroalkoxy and C₁₋₃alkoxy. In someembodiments, Cy² is unsubstituted or substituted with 1-2 substituentsindependently selected from Cl, F, NHCH₃, N(CH₃)₂, CF₃, CH₃, CH₃CH₂,(CH₃)₂CH₂, CH₃O, CH₃CH₂O, (CH₃)₂CH₂O, CF₃O and CF₃O.

In some embodiments, the compound of Formula I is selected fromcompounds I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-10, I-11, I-12,I-13, I-14, I-15, I-16, I-17, I-18, I-19, I-20, I-21, I-22, I-23, I-24,I-25, I-26, I-27, I-28, I-29, I-30, I-31, I-32, I-33, I-34, I-35, I-36,I-37, I-38, I-39, I-40, I-41, I-42, I-43, I-44, I-45, I-46 and I-47 asshown in Table 2, or a pharmaceutically acceptable salt, solvate and/orprodrug thereof.

In an embodiment, the compounds of the application are in the form of apharmaceutically acceptable salt.

The compounds of the present application are suitably formulated in aconventional manner into compositions using one or more carriers.Accordingly, the present application also includes a compositioncomprising one or more compounds of the application and a carrier. Thecompounds of the application are suitably formulated into pharmaceuticalcompositions for administration to subjects in a biologically compatibleform suitable for administration in vivo. Accordingly, the presentapplication further includes a pharmaceutical composition comprising oneor more compounds of the application and a pharmaceutically acceptablecarrier. In embodiments of the application the pharmaceuticalcompositions are used in the treatment of nay of the diseases, disordersor conditions described herein.

The compounds of the application are administered to a subject in avariety of forms depending on the selected route of administration, aswill be understood by those skilled in the art. For example, a compoundof the application is administered by oral, inhalation, parenteral,buccal, sublingual, nasal, rectal, vaginal, patch, pump, topical ortransdermal administration and the pharmaceutical compositionsformulated accordingly. In some embodiments, administration is by meansof a pump for periodic or continuous delivery. Conventional proceduresand ingredients for the selection and preparation of suitablecompositions are described, for example, in Remington's PharmaceuticalSciences (2000-20th edition) and in The United States Pharmacopeia: TheNational Formulary (USP 24 NF19) published in 1999.

Parenteral administration includes systemic delivery routes other thanthe gastrointestinal (GI) tract, and includes, for example intravenous,intra-arterial, intraperitoneal, subcutaneous, intramuscular,transepithelial, nasal, intrapulmonary (for example, by use of anaerosol), intrathecal, rectal and topical (including the use of a patchor other transdermal delivery device) modes of administration.Parenteral administration may be by continuous infusion over a selectedperiod of time.

In some embodiments, a compound of the application is orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or it is enclosed in hard or soft shell gelatincapsules, or it is compressed into tablets, or it is incorporateddirectly with the food of the diet. In some embodiments, the compound isincorporated with excipient and used in the form of ingestible tablets,buccal tablets, troches, capsules, caplets, pellets, granules, lozenges,chewing gum, powders, syrups, elixirs, wafers, aqueous solutions andsuspensions, and the like. In the case of tablets, carriers that areused include lactose, corn starch, sodium citrate and salts ofphosphoric acid. Pharmaceutically acceptable excipients include bindingagents (e.g., pregelatinized maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium phosphate); lubricants (e.g., magnesium stearate,talc or silica); disintegrants (e.g., potato starch or sodium starchglycolate); or wetting agents (e.g., sodium lauryl sulphate). Inembodiments, the tablets are coated by methods well known in the art. Inthe case of tablets, capsules, caplets, pellets or granules for oraladministration, pH sensitive enteric coatings, such as Eudragits™designed to control the release of active ingredients are optionallyused. Oral dosage forms also include modified release, for exampleimmediate release and timed-release, formulations. Examples ofmodified-release formulations include, for example, sustained-release(SR), extended-release (ER, XR, or XL), time-release or timed-release,controlled-release (CR), or continuous-release (CR or Contin), employed,for example, in the form of a coated tablet, an osmotic delivery device,a coated capsule, a microencapsulated microsphere, an agglomeratedparticle, e.g., as of molecular sieving type particles, or, a finehollow permeable fiber bundle, or chopped hollow permeable fibers,agglomerated or held in a fibrous packet. Timed-release compositions areformulated, for example as liposomes or those wherein the activecompound is protected with differentially degradable coatings, such asby microencapsulation, multiple coatings, etc. Liposome delivery systemsinclude, for example, small unilamellar vesicles, large unilamellarvesicles and multilamellar vesicles. In some embodiments, liposomes areformed from a variety of phospholipids, such as cholesterol,stearylamine or phosphatidylcholines. For oral administration in acapsule form, useful carriers or diluents include lactose and dried cornstarch.

In some embodiments, liquid preparations for oral administration takethe form of, for example, solutions, syrups or suspensions, or they aresuitably presented as a dry product for constitution with water or othersuitable vehicle before use. When aqueous suspensions and/or emulsionsare administered orally, the compound of the application is suitablysuspended or dissolved in an oily phase that is combined withemulsifying and/or suspending agents. If desired, certain sweeteningand/or flavoring and/or coloring agents are added. Such liquidpreparations for oral administration are prepared by conventional meanswith pharmaceutically acceptable additives such as suspending agents(e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters or ethyl alcohol); and preservatives(e.g., methyl or propyl p-hydroxybenzoates or sorbic acid). Usefuldiluents include lactose and high molecular weight polyethylene glycols.

It is also possible to freeze-dry the compounds of the application anduse the lyophilizates obtained, for example, for the preparation ofproducts for injection.

In some embodiments, a compound of the application is administeredparenterally. For example, solutions of a compound of the applicationare prepared in water suitably mixed with a surfactant such ashydroxypropylcellulose. In some embodiments, dispersions are prepared inglycerol, liquid polyethylene glycols, DMSO and mixtures thereof with orwithout alcohol, and in oils. Under ordinary conditions of storage anduse, these preparations contain a preservative to prevent the growth ofmicroorganisms. A person skilled in the art would know how to preparesuitable formulations. For parenteral administration, sterile solutionsof the compounds of the application are usually prepared, and the pH'sof the solutions are suitably adjusted and buffered. For intravenoususe, the total concentration of solutes should be controlled to renderthe preparation isotonic. For ocular administration, ointments ordroppable liquids are delivered, for example, by ocular delivery systemsknown to the art such as applicators or eye droppers. In someembodiment, such compositions include mucomimetics such as hyaluronicacid, chondroitin sulfate, hydroxypropyl methylcellulose or polyvinylalcohol, preservatives such as sorbic acid, EDTA or benzyl chromiumchloride, and the usual quantities of diluents or carriers. Forpulmonary administration, diluents or carriers will be selected to beappropriate to allow the formation of an aerosol.

In some embodiments, a compound of the application is formulated forparenteral administration by injection, including using conventionalcatheterization techniques or infusion. Formulations for injection are,for example, presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. In some embodiments,the compositions take such forms as sterile suspensions, solutions oremulsions in oily or aqueous vehicles, and contain formulating agentssuch as suspending, stabilizing and/or dispersing agents. In all cases,the form must be sterile and must be fluid to the extent that easysyringability exists. Alternatively, the compounds of the applicationare suitably in a sterile powder form for reconstitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

In some embodiments, compositions for nasal administration areconveniently formulated as aerosols, drops, gels or powders. Forintranasal administration or administration by inhalation, the compoundsof the application are conveniently delivered in the form of a solution,dry powder formulation or suspension from a pump spray container that issqueezed or pumped by the patient or as an aerosol spray presentationfrom a pressurized container or a nebulizer. Aerosol formulationstypically comprise a solution or fine suspension of the active substancein a physiologically acceptable aqueous or non-aqueous solvent and areusually presented in single or multidose quantities in sterile form in asealed container, which, for example, take the form of a cartridge orrefill for use with an atomising device. Alternatively, the sealedcontainer is a unitary dispensing device such as a single dose nasalinhaler or an aerosol dispenser fitted with a metering valve which isintended for disposal after use. Where the dosage form comprises anaerosol dispenser, it will contain a propellant which is, for example, acompressed gas such as compressed air or an organic propellant such asfluorochlorohydrocarbon. Suitable propellants include but are notlimited to dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, heptafluoroalkanes, carbon dioxide and othersuitable gases. In the case of a pressurized aerosol, the dosage unit issuitably determined by providing a valve to deliver a metered amount. Insome embodiments, the pressurized container or nebulizer contains asolution or suspension of the active compound. Capsules and cartridges(made, for example, from gelatin) for use in an inhaler or insufflatorare, for example, formulated containing a powder mix of a compound ofthe application and a suitable powder base such as lactose or starch.The aerosol dosage forms can also take the form of a pump-atomizer.

Compositions suitable for buccal or sublingual administration includetablets, lozenges, and pastilles, wherein a compound of the applicationis formulated with a carrier such as sugar, acacia, tragacanth, orgelatin and glycerine. Compositions for rectal administration areconveniently in the form of suppositories containing a conventionalsuppository base such as cocoa butter.

Suppository forms of the compounds of the application are useful forvaginal, urethral and rectal administrations. Such suppositories willgenerally be constructed of a mixture of substances that is solid atroom temperature but melts at body temperature. The substances commonlyused to create such vehicles include but are not limited to theobromaoil (also known as cocoa butter), glycerinated gelatin, otherglycerides, hydrogenated vegetable oils, mixtures of polyethyleneglycols of various molecular weights and fatty acid esters ofpolyethylene glycol. See, for example: Remington's PharmaceuticalSciences, 16th Ed., Mack Publishing, Easton, Pa., 1980, pp. 1530-1533for further discussion of suppository dosage forms.

In some embodiments a compound of the application is coupled withsoluble polymers as targetable drug carriers. Such polymers include, forexample, polyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamide-phenol,polyhydroxy-ethylaspartamide-phenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, in some embodiments, acompound of the application is coupled to a class of biodegradablepolymers useful in achieving controlled release of a drug, for example,polylactic acid, polyglycolic acid, copolymers of polylactic andpolyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates andcrosslinked or amphipathic block copolymers of hydrogels.

A compound of the application, including pharmaceutically acceptablesalts, solvates and/or prodrugs thereof, is suitably used on their ownbut will generally be administered in the form of a pharmaceuticalcomposition in which the one or more compounds of the application (theactive ingredient) is in association with a pharmaceutically acceptablecarrier. Depending on the mode of administration, the pharmaceuticalcomposition will comprise from about 0.05 wt % to about 99 wt % or about0.10 wt % to about 70 wt %, of the active ingredient, and from about 1wt % to about 99.95 wt % or about 30 wt % to about 99.90 wt % of apharmaceutically acceptable carrier, all percentages by weight beingbased on the total composition.

III. Methods and Uses of the Application

The compounds of the application have been shown to be selectiveinhibitors of hGGPPS activity. In some embodiments, compounds of theapplication are more selective in inhibiting hGGPPS activity that hFPPSactivity. In some embodiments, additional (albeit weaker) activity ininhibiting hFPPS is not detrimental to the proposed therapeutic value ofthese compounds, but may be of an additional benefit.

The present application includes a method for inhibiting hGGPPS activityin a cell, either in a biological sample or in a patient, comprisingadministering an effective amount of one or more compounds of theapplication to the cell. The application also includes a use of one ormore compounds of the application for inhibition of hGGPPS activity in acell as well as a use of one or more compounds of the application forthe preparation of a medicament for inhibition of hGGPPS activity in acell. The application further includes one or more compounds of theapplication for use in inhibiting hGGPPS activity in a cell.

The present application includes a method for inhibitinggeranylgeranylation of proteins in a cell, either in a biological sampleor in a patient, comprising administering an effective amount of one ormore compounds of the application to the cell. The application alsoincludes a use of one or more compounds of the application forinhibition of geranylgeranylation in a cell as well as a use of one ormore compounds of the application for the preparation of a medicamentfor inhibition of geranylgeranylation of proteins in a cell. Theapplication further includes one or more compounds of the applicationfor use in inhibiting geranylgeranylation of proteins in a cell.

As the compounds of the application have been shown to be capable ofinhibiting hGGPPS activity, the compounds of the application are usefulfor treating diseases, disorders or conditions mediated by hGGPPS.Therefore the compounds of the present application are useful asmedicaments. Accordingly, the present application includes a compound ofthe application for use as a medicament.

As the compounds of the application have been shown to also be capableof inhibiting hFPPS activity, in some embodiments, the compounds of theapplication are useful for treating diseases, disorders or conditionsmediated by hFPPS. Therefore the compounds of the present applicationcan block both farnesylation and geranylgeranylation of proteins andconsequently, are useful as medicaments as such.

The present application also includes a method of treating a disease,disorder or condition mediated by hGGPPS comprising administering atherapeutically effective amount of one or more compounds of theapplication to a subject in need thereof.

The present application also includes a use of one or more compounds ofthe application for treatment of a disease, disorder or conditionmediated by hGGPPS, or treatable by inhibition of geranylgeranylation ofproteins, as well as a use of one or more compounds of the applicationfor the preparation of a medicament for treatment of a disease, disorderor condition mediated by hGGPPS, or treatable by inhibition ofgeranylgeranylation of proteins. The application further includes one ormore compounds of the application for use in treating a disease,disorder or condition mediated by hGGPPS, or treatable by inhibition ofgeranylgeranylation of proteins.

In an embodiment, the disease, disorder or condition mediated by hGGPPS,or treatable by inhibition of geranylgeranylation of proteins, is aneoplastic disorder. Accordingly, the present application also includesa method of treating a neoplastic disorder comprising administering atherapeutically effective amount of one or more compounds of theapplication to a subject in need thereof. The present application alsoincludes a use of one or more compounds of the application for treatmentof a neoplastic disorder as well as a use of one or more compounds ofthe application for the preparation of a medicament for treatment of aneoplastic disorder. The application further includes one or morecompounds of the application for use in treating a neoplastic disorder.In an embodiment, the treatment is in an amount effective to ameliorateat least one symptom of the neoplastic disorder, for example, reducedcell proliferation, reduced tumor mass, or reduce lytic bone disease,among others, in a subject in need of such treatment.

In another embodiment of the present application, the disease, disorderor condition mediated by hGGPPS, or treatable by inhibition ofgeranylgeranylation of proteins, is cancer. Accordingly, the presentapplication also includes a method of treating cancer comprisingadministering a therapeutically effective amount of one or morecompounds of the application to a subject in need thereof. The presentapplication also includes a use of one or more compounds of theapplication for treatment of cancer as well as a use of one or morecompounds of the application for the preparation of a medicament fortreatment of cancer. The application further includes one or morecompounds of the application for use in treating cancer. In anembodiment, the compound is administered for the prevention of cancer ina subject such as a mammal having a predisposition for cancer.

In view of the inhibition of hFPPS activity by the compounds of theapplication, in another embodiment, the disease, disorder or conditionis also one that is mediated by hFPPS, or treatable by inhibition offarnesylation. In an embodiment, this disease is cancer.

In some embodiments, the cancer is selected from hematological cancerssuch as multiple myeloma, chronic myelogenous leukemia and acutemonocytic leukemia. Metastases of the aforementioned cancers can also betreated in accordance with the methods described herein.

In some embodiments, the cancer is selected from solid tumor cancerssuch as ovarian cancer, including those expressing multidrug resistance,pancreatic, fibrosarcoma, colorectal, brain and non-small cell lungcancers. Metastases of the aforementioned cancers can also be treated inaccordance with the methods described herein.

In some embodiments, the disease, disorder or condition mediated byhGGPPS, or treatable by inhibition of geranylgeranylation of proteins,is osteoporosis or cancer-related lytic bone disease.

In an embodiment, the hGGPPS-mediated disease, disorder or condition isa disease, disorder or condition associated with an uncontrolled and/orabnormal cellular activity affected directly or indirectly by hGGPPS. Inanother embodiment, the uncontrolled and/or abnormal cellular activitythat is affected directly or indirectly by hGGPPS is proliferativeactivity in a cell. Accordingly, the application also includes a methodof inhibiting proliferative activity in a cell, comprising administeringan effective amount of one or more compounds of the application to thecell. The present application also includes a use of one or morecompounds of the application for inhibition of proliferative activity ina cell as well as a use of one or more compounds of the application forthe preparation of a medicament for inhibition of proliferative activityin a cell. The application further includes one or more compounds of theapplication for use in inhibiting proliferative activity in a cell.

In an embodiment, the hFPPS-mediated disease, disorder or condition is adisease, disorder or condition associated with an uncontrolled and/orabnormal cellular activity affected directly or indirectly by hFPPS. Inanother embodiment, the uncontrolled and/or abnormal cellular activitythat is affected directly or indirectly by hFPPS is proliferativeactivity in a cell.

The present application also includes a method of inhibitinguncontrolled and/or abnormal cellular activities mediated directly orindirectly by hGGPPS in a cell, either in a biological sample or in asubject, comprising administering an effective amount of one or morecompounds of the application to the cell. The application also includesa use of one or more compounds of the application for inhibition ofuncontrolled and/or abnormal cellular activities mediated directly orindirectly by hGGPPS in a cell as well as a use of one or more compoundsof the application for the preparation of a medicament for inhibition ofuncontrolled and/or abnormal cellular activities mediated directly orindirectly by hGGPPS in a cell. The application further includes one ormore compounds of the application for use in inhibiting uncontrolledand/or abnormal cellular activities mediated directly or indirectly byhGGPPS in a cell.

The present application also includes a method of inhibitinguncontrolled and/or abnormal cellular activities mediated directly orindirectly by hFPPS in a cell, either in a biological sample or in asubject, comprising administering an effective amount of one or morecompounds of the application to the cell. The application also includesa use of one or more compounds of the application for inhibition ofuncontrolled and/or abnormal cellular activities mediated directly orindirectly by hFPPS in a cell as well as a use of one or more compoundsof the application for the preparation of a medicament for inhibition ofuncontrolled and/or abnormal cellular activities mediated directly orindirectly by hFPPS in a cell. The application further includes one ormore compounds of the application for use in inhibiting uncontrolledand/or abnormal cellular activities mediated directly or indirectly byhFPPS in a cell.

The present application also includes a method of treating a disease,disorder or condition that is mediated by hGGPPS, or treatable byinhibition of geranylgeranylation of proteins, comprising administeringa therapeutically effective amount of one or more compounds of theapplication in combination with another known agent useful for treatmentof a disease, disorder or condition mediated by hGGPPS, or treatable byinhibition of geranylgeranylation, to a subject in need thereof. Thepresent application also includes a use of one or more compounds of theapplication in combination with a known agent useful for treatment of adisease, disorder or condition mediated by hGGPPS, or treatable byinhibition of geranylgeranylation, for treatment of a disease, disorderor condition mediated by hGGPPS.

The present application also includes a method of treating a disease,disorder or condition that is mediated by hFPPS, or treatable byinhibition of farnesylation of proteins, comprising administering atherapeutically effective amount of one or more compounds of theapplication in combination with another known agent useful for treatmentof a disease, disorder or condition mediated by hFPPS, or treatable byinhibition of farnesylation, to a subject in need thereof. The presentapplication also includes a use of one or more compounds of theapplication in combination with a known agent useful for treatment of adisease, disorder or condition mediated by hFPPS, or treatable byinhibition of farnesylation, for treatment of a disease, disorder orcondition mediated by hFPPS.

In a further embodiment, the disease, disorder or condition mediatedhGGPPS, or treatable by inhibition of geranylgeranylation of proteins,and/or that is mediated by hFPPS, or treatable by inhibition offarnesylation of proteins, is cancer and the one or more compounds ofthe application are administered in combination with one or moreadditional cancer treatments. In another embodiment, the additionalcancer treatment is selected from radiotherapy, chemotherapy, targetedtherapies such as antibody therapies and small molecule therapies suchas tyrosine-kinase inhibitors, immunotherapy, hormonal therapy andanti-angiogenic therapies.

In another embodiment of the present application, the disease, disorderor condition that has been implicated with upregulation of hGGPPSactivity, and consequently, may be treatable by inhibition ofgeranylgeranylation, is Alzheimer's Disease. Accordingly, the presentapplication also includes a method of treating taupathies that areassociated with neurodegeneration, such as Alzheimer's Disease,comprising administering a therapeutically effective amount of one ormore compounds of the application to a subject in need thereof. Thepresent application also includes a use of one or more compounds of theapplication for the treatment of taupathies that are associated withneurodegeneration, such as Alzheimer's Disease, as well as a use of oneor more compounds of the application for the preparation of a medicamentfor the treatment of taupathies that are associated withneurodegeneration, such as Alzheimer's Disease. The application furtherincludes one or more compounds of the application for use in thetreatment of taupathies that are associated with neurodegeneration, suchas Alzheimer's Disease. In an embodiment, the compound is administeredor used for the prevention of neurofibrillary tangles induced by highlevels of phosphorylated tau protein in the brain which is stronglyassociated with Alzheimer's Disease in a subject such as a mammal havinga predisposition for Alzheimer's Disease.

A compound of the application is either used alone or in combinationwith other known agents useful for treating diseases, disorders orconditions that are mediated by hGGPPS, and those that are treatablewith an hGGPPS inhibitor and/or with other known agents useful fortreating diseases, disorders or conditions that are mediated by hFPPS,and those that are treatable with an hFPPS inhibitor. When used incombination with other agents useful in treating diseases, disorders orconditions mediated by hGGPPS inhibition and/or hFPPS inhibition, it isan embodiment that a compound of the application is administeredcontemporaneously with those agents. As used herein, “contemporaneousadministration” of two substances to a subject means providing each ofthe two substances so that they are both active in the individual at thesame time. The exact details of the administration will depend on thepharmacokinetics of the two substances in the presence of each other,and can include administering the two substances within a few hours ofeach other, or even administering one substance within 24 hours ofadministration of the other, if the pharmacokinetics are suitable.Design of suitable dosing regimens is routine for one skilled in theart. In particular embodiments, two substances will be administeredsubstantially simultaneously, i.e., within minutes of each other, or ina single composition that contains both substances. It is a furtherembodiment of the present application that a combination of agents isadministered to a subject in a non-contemporaneous fashion. In anembodiment, a compound of the present application is administered withanother therapeutic agent simultaneously or sequentially in separateunit dosage forms or together in a single unit dosage form. Accordingly,the present application provides a single unit dosage form comprisingone or more compounds of the application, an additional therapeuticagent, and a pharmaceutically acceptable carrier.

Thus the methods of the present application are applicable to both humantherapy and veterinary applications. In an embodiment, the subject is amammal. In another embodiment, the subject is human.

The dosage of a compound of the application varies depending on manyfactors such as the pharmacodynamic properties of the compound, the modeof administration, the age, health and weight of the recipient, thenature and extent of the symptoms, the frequency of the treatment andthe type of concurrent treatment, if any, and the clearance rate of thecompound in the subject to be treated. One of skill in the art candetermine the appropriate dosage based on the above factors. In someembodiments, a compound of the application is administered initially ina suitable dosage that is adjusted as required, depending on theclinical response. Dosages will generally be selected to maintain aserum level of the compound of the application from about 0.01 μg/cc toabout 1000 μg/cc, or about 0.1 μg/cc to about 100 μg/cc. As arepresentative example, oral dosages of one or more compounds of theapplication will range between about 1 mg per day to about 2000 mg perday for an adult, about 1 mg per day to about 1000 mg per day, suitablyabout 1 mg per day to about 500 mg per day, more suitably about 1 mg perday to about 200 mg per day. For parenteral administration, arepresentative amount is from about 0.001 mg/kg to about 10 mg/kg, about0.01 mg/kg to about 10 mg/kg, about 0.01 mg/kg to about 1 mg/kg or about0.1 mg/kg to about 1 mg/kg will be administered. For oraladministration, a representative amount is from about 0.001 mg/kg toabout 10 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.01 mg/kg toabout 1 mg/kg or about 0.1 mg/kg to about 1 mg/kg. For administration insuppository form, a representative amount is from about 0.1 mg/kg toabout 10 mg/kg or about 0.1 mg/kg to about 1 mg/kg. In an embodiment ofthe application, compositions are formulated for oral administration andthe one or more compounds are suitably in the form of tablets containing0.25, 0.5, 0.75, 1.0, 5.0, 10.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0,70.0, 75.0, 80.0, 90.0, 100.0, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mg of activeingredient per tablet. In embodiments of the application the one or morecompounds of the application are administered in a single daily, weeklyor monthly dose or the total daily dose is divided into two, three orfour daily doses.

In the above, the term “a compound” also includes embodiments whereinone or more compounds are referenced.

IV. Methods of Preparing Compounds of the Application

Compounds of the present application are prepared by various syntheticprocesses. The choice of particular structural features and/orsubstituents may influence the selection of one process over another.The selection of a particular process to prepare a given compound of theapplication is within the purview of the person of skill in the art.Some starting materials for preparing compounds of the presentapplication are available from commercial chemical sources. Otherstarting materials, for example as described below, are readily preparedfrom available precursors using straightforward transformations that arewell known in the art.

In some embodiments of the application, compounds of Formula I that arebased on a thieno[2,3-d]pyrimidin-4-amine scaffold, wherein X is NH andR¹ is a bisphosphonate or bisphosphonate ester, are prepared as shown inSchemes 1, 6 and 7. In some embodiments, the thiophene intermediate 1 isprepared from the commercially available dimer of mercaptoacetaldehyde,1,4-dithiane-2,5-diol or from an aldehyde as previously reported [seefor example: (a) Leung, et al. J. Med. Chem. 2013, 56, 7939-7950. (b)Leung, et al Bioorg. Med. Chem. 2013, 21, 2229-2240. (c) Gao, et al.Bioorg. Med. Chem. Lett. 2013, 23, 1953-1956]. In some embodiments, asshown in Scheme 1, the 2-amino-3-cyanothiophene core 1 is elaborated tosubstituted 4a,7a-dihydrothieno[2,3-d]pyrimidin-4-amine core of generalstructure 3 via direct cyclization with various aryl and or heteroarylnitriles in the presence of a base (Chen, et. al. Synthesis 2010, 14,2413-2418). In some embodiments, as shown in Scheme 1, intermediate 1 iscyclized to the thioether intermediate 2 having a methyl thioethermoiety at the C-2 of the pyrimidine ring, by condensing 1 with methylthiocyanate under acidic conditions (Barbay, et. al. WO 2010/045006 A1).In further embodiments, upon introduction of the bisphosphonatetetraesters (i.e. conversion of 2 to 4; R^(a)=SMe), the thioetherintermediate 4 is suitable for the Liebeskind-Srogl cross-couplingreaction (Liebeskind and Srogl Org. Lett., 2002, 4, 979-981; Barbay, et.al. WO 2010/045006 A1) with various boronic acids to give compounds 5with significant structural diversity at R^(a), including various aryland heteroaryl groups. In some embodiments, intermediates 5 includeanalogs where R^(a) is an aromatic or heteroaromatic group with a nirosubstituent, for example a nitrophenyl (e.g. 5a in Scheme 6) or an aryl,heteroaryl or other heterocycle with a carboxylic acid substituent (e.g.5b in Scheme 6). In some embodiments, the latter compounds are furtherelaborated to analogs such as compounds having an amide, sulfonamide orurea linker L between the two groups Cy¹ and Cy² as shown in Schemes 6and 7. In some embodiments, the thioether moiety of intermediates 2 canbe oxidized to the corresponding methylsulfonyl intermediates withm-CPBA, followed by nucleophic displacement of the methylsulfonyl moietyby S_(N)Ar to give compounds with general formula 7, which can befurther elaborated to hGGPPS inhibitors with general structure 8, asshown in Scheme 1. It would be known to those skilled in the art oforganic synthesis that the R^(e) moiety of inhibitors 8 could also beprepared in several steps, starting from an intermediate of 7 havingR^(e) as an appropriate functional group, including, but not limited toan amine, a carboxylic acid, an acid chloride, a halogen or an alcohol,which can be further elaborated using similar synthetic methodologiesfor example to those shown in Schemes 6 and 7 for the conversion ofintermediates 5a, 5b and 5c of Scheme 6 to the final inhibitors, forexample, analogs Ia, Ib, Ic and Id. In Schemes 1, 5 and 6, the variablesQ and W represent substituted or unsubstituted carbon or nitrogen, or aheteroatom selected from O and S, q is 0, 1, 2, 3 or 4 and R^(a)-R^(e)represent various functional groups that are found in Formula I or canbe converted using known methods, to functional groups found in FormulaI.

It would be known to those skilled in the art of organic synthesis thatsimilar synthetic methodologies to those described in Schemes 1, 6 and 7for the preparation of thieno[2,3-d]pyrimidine-based compounds Formula Ican also be used to prepare analogs with a thieno[3,2-d]pyrimidine-basedcore. Examples of the synthesis of the building blocks for thepreparation of these compounds are shown in Scheme 2. In Scheme 2, thevariables Q and W represent substituted or unsubstituted carbon ornitrogen, or a heteroatom selected from O and S, q is 0, 1, 2, 3 or 4and R^(e) represents various functional groups that are found in FormulaI or can be converted using known methods, to functional groups found inFormula I.

It would be known to those skilled in the art of organic synthesis thatsimilar synthetic methodologies to those described in Schemes 1, 2, 6and 7 for the preparation of thienopyrimidine-based compounds of FormulaI can also be used to prepare analogs that arm thiazolopyrimidine-basedcompounds of Formula I. Examples of the synthesis of building blocks forthe preparation of thiazolo[5,4-d]pyrimidine-based compounds are shownin Scheme 3.

It would be known to those skilled in the art of organic synthesis thatsimilar synthetic methodologies to those described in Schemes 1, 2, 3, 6and 7 for the preparation of thienopyrimidine-based andthiazolopyrimidine-based compounds of Formula I can also be used toprepare purine-based compounds of Formula I. Examples of the synthesisof building blocks for the preparation of these compounds are shown inScheme 4.

Similar synthetic methodologies to those described in the Schemes 1, 2,3, 4, 6 and 7 for the preparation of thienopyrimidine-based,thiazolopyrimidine-based and purine-based compounds of Formula I canalso be used to prepare pyrrolopyrimidine-based compounds of Formula I.As an example, the synthesis of building blocks for the preparation of7H-pyrrolo[2,3-d]pyrimidine-based inhibitors is shown in Scheme 5.

Synthetic methodologies for the preparation of furopyrimidine-basedcompounds of Formula I starting from the dichloro intermediates that arestructurally equivalent to compound 34 (in Scheme 5), but where thenitrogen of the 5-membered pyrrole ring is replaced by an oxygen atom,are known (see for example WO 2008/073785 and Roecker et al. Biorg. Med.Chem. Lett. 2014, 24, 2079-2085). These intermediates can be furtherelaborated to final compounds of Formula I using the syntheticmethodologies described in the Schemes 4, 5, 6 and 7. Similarly, thesynthesis of oxazolopyrimidiners can be achieved using previouslyreported synthetic intermediates. For example an intermediate such asthe 5,7-dichlorooxazolo[5,4-d]pyrimidine 37 shown in Scheme 7 (for anexample of the synthesis, see WO 2009/013545) can be further elaboratedto final compound of Formula I using the synthetic methodologiesdescribed in Schemes 4, 5, 6 and 7. In some embodiments, compounds ofFormula I are synthesized, for example through direct connection of twogroups Cy¹ and Cy² or the incorporation of a linker between group Cy¹and Cy², which in some embodiments is a direct bond, methylene, anamide, a reversed amide, a sulfonamide, or a urea (Scheme 7). Forexample, in some embodiments, the nitro group of 5a in Scheme 6 is firstreduced to the amine using tin (II) chloride and the aniline product 5dis then coupled with an acyl chloride or a carboxylic acid usingstandard peptide chemistry to give analogs with general structure Ic, orwith a sulfonyl chloride to give analogs Id, or with an isocyanate togive analogs with a urea linker L. Similarly, in some embodimentscompounds of Formula I are prepared from the carbocylic acid 5b (Scheme6) or via Liebeskind-Srogl cross-coupling reaction between 4 (Scheme 1)and appropriate boronic acid to give eventually the reversed amidederivatives Ib. In some embodiments, compounds of Formula I are preparedvia Pd-catalyzed cross-coupling reactions with the aryl halides (5c,Scheme 6) to give biaryl derivatives Ia.

In some embodiments, compounds of Formula I arethiazolo[5,4-d]pyrimidine-based, 9H-purine-based and7H-pyrrolo[2,3-d]pyrimidine-based bisphosphonates prepared usingprotocols similar to those described above from the respective dichlorointermediates 21 (Scheme 3), 27 (Scheme 4) and 34 (Scheme 5) and brieflysummarized in Scheme 7.

In Schemes 6 and 7, the variables Q and W represent substituted orunsubstituted carbon or nitrogen, or a heteroatom selected from O and S,q is 0, 1, 2, 3 or 4 and R^(e) represents various functional groups thatare found in Formula I or can be converted using known methods, tofunctional groups found in Formula I.

A person skilled in the art of organic synthesis would understand thatthe procedures summarized in Schemes 1-7, for the preparation of FormulaI compounds that are thienopyrimidine-, thiazolopyrimidine-, purine-,pyrrolopyrimidine-, furopyrimidine- or oxazolopyrimidine-based compoundswith general formulas Ia, Ib, Ic, Id, Ie, If, Ig, Ih, Ii and Ij can beprepared from other key intermediates that can be accessed frompublished procedures. In some embodiments the main starting materialsare obtained from a commercial source or are prepared using theliterature protocols provided in the synthesis of specific Examplesbelow. In some embodiments, compounds of formula 21, 27, 34, 35 and 37can be further elaborated to Examples Ie to Ij via first nucleophilicaromatic substitution (S_(N)Ar) reactions of the chloride at the C-4carbon of the pyrimidine ring with ammonia or a suitable protected amineto give intermediates such as 22 (in Scheme 3), 30 (in Scheme 4), 36 (inScheme 5), followed by metal-catalyzed cross-coupling reactions orS_(N)Ar displacement of the C-2 chloro moiety. For instance, a varietyof aryl- or heteroaryl-boronic acids are reacted with intermediates 22,30 or 36 via Suzuki cross-coupling reaction. Additionally, in someembodiments, Buchwald-Hartwig amination is done to prepare a diverselibrary of compounds varied at the position indicated. Othermetal-mediated cross coupling reactions that would be known to thoseskilled in organic synthesis can also be used to increase the structuraldiversity of compounds with general structure of Formula I.

Throughout the processes described herein it is to be understood that,where appropriate, suitable protecting groups will be added to, andsubsequently removed from, the various reactants and intermediates in amanner that will be readily understood by one skilled in the art.Conventional procedures for using such protecting groups as well asexamples of suitable protecting groups are described, for example, in“Protective Groups in Organic Synthesis”, T. W. Green, P. G. M. Wuts,Wiley-Interscience, New York, (4^(th) Edition, 2007). It is also to beunderstood that a transformation of a group or substituent into anothergroup or substituent by chemical manipulation is conducted on anyintermediate or final product on the synthetic path toward the finalproduct, in which the possible type of transformation is limited only byinherent incompatibility of other functionalities carried by themolecule at that stage to the conditions or reagents employed in thetransformation. Such inherent incompatibilities, and ways to circumventthem by carrying out appropriate transformations and synthetic steps ina suitable order, will be readily understood to one skilled in the art.Examples of transformations are given herein, and it is to be understoodthat the described transformations are not limited only to the genericgroups or substituents for which the transformations are exemplified.References and descriptions of other suitable transformations are givenin “Comprehensive Organic Transformations—A Guide to Functional GroupPreparations” R. C. Larock, VHC Publishers, Inc. (1989). References anddescriptions of other suitable reactions are described in textbooks oforganic chemistry, for example, “Advanced Organic Chemistry”, March, 4thed. McGraw Hill (1992) or, “Organic Synthesis”, Smith, McGraw Hill,(1994). Techniques for purification of intermediates and final productsinclude, for example, straight and reversed phase chromatography oncolumn or rotating plate, recrystallisation, distillation andliquid-liquid or solid-liquid extraction, which will be readilyunderstood by one skilled in the art.

All process/method steps described herein are to be conducted underconditions sufficient to provide the desired product. A person skilledin the art would understand that all reaction conditions, including, forexample, reaction solvent, reaction time, reaction temperature, reactionpressure, reactant ratio and whether or not the reaction should beperformed under an anhydrous or inert atmosphere, can be varied tooptimize the yield of the desired product and it is within their skillto do so.

The following non-limiting examples are illustrative of the presentapplication:

EXAMPLES

Chemicals and solvents were purchased from commercial suppliers and usedwithout further purification. Normal phase column chromatography onsilica gel was performed using a CombiFlash instrument using the solventgradient, as indicated. Reverse phase preparative HPLC was carried outusing a Waters Atlantis Prep T3 OBD C18 5 μm 19×50 mm column; Solvent A:H₂O, 0.1% formic acid; Solvent B: CH₃CN, 0.1% formic acid; Mobile phase:gradient from 95% A and 5% B to 5% A and 95% B in 17 min acquisitiontime; flow rate: 1 mL/min. The homogeneity of final compounds wasconfirmed to be ≥95% by reversed-phase HPLC using a Waters ALLIANCE®instrument (e2695 with 2489 UV detector, 3100 mass spectrometer, C18 5μm column): Solvent A: H₂O, 0.1% formic acid; Solvent B: CH₃CN, 0.1%formic acid; Mobile phase: linear gradient from 95% A and 5% B to 0% Aand 100% B in 13 mins. Key compounds were fully characterized by ¹H,¹³C, ³¹P NMR and MS and HRMS. Chemical shifts (δ) are reported in ppmrelative to the internal deuterated solvent. The NMR spectra of allfinal bisphosphonate compounds were acquired in D₂O (either afterconversion to their corresponding tri-sodium salt or by addition of ˜2%ND₄OD). In some cases, the Cα to the bisphosphonate was broad andoverlapped with the solvent peak, as confirmed by HSQC. The highresolution MS spectra of final products were recorded using electrosprayionization (ESI^(+/−)) and Fourier transform ion cyclotron resonancemass analyzer (FTMS).

Example 1: Preparation of Compounds Synthesis of2-(methylthio)thieno[2,3-d]pyrimidin-4-amine (2, R=H) (Scheme 1)

2-aminothiophene-3-carbonitrile (1, R=H; 1.0 eq) was added to HCl indioxane (4M; 6.0 eq), followed by methyl thiocyanate (1.0 eq). Theresulting suspension was heated to 70° C. in a sealed pressure tube for24 h. The mixture was allowed to cool to rt and the resulting greenprecipitate was collected by vacuum filtration. The solid was dissolvedin EtOAc and washed with saturated aqueous NaHCO₃. The layers wereseparated and the aqueous phase was extracted further with EtOAc. Theorganic extracts were combined, washed with brine, dried over Na₂SO₄,and concentrated in vacuo. Product was obtained as a light brown solid(typical isolated yield was between 60%-80%) and used in the next stepwithout further purification. ¹H NMR (500 MHz, DMSO-d₆) δ 7.55 (br_s,2H), 7.47 (d, J=5.9 Hz, 1H), 7.36 (d, J=5.9 Hz, 1H), 2.46 (s, 3H). ¹³CNMR (126 MHz, DMSO-d₆) δ 167.5, 166.5, 158.3, 120.7, 120.2, 113.4, 13.8.MS [ESI⁺] m/z: 198.0 [M+H]⁺.

Tetraethyl(((2-(methylthio)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)(4, R=H) (Scheme 1)

A pressure vessel charged with2-(methylthio)thieno[2,3-d]pyrimidin-4-amine (2, R=H; 1.0 eq) in drytoluene (1.0 M) was added with diethyl phosphite (7.0 eq) and triethylorthoformate (1.7 eq). The mixture was heated at 130° C. for 40 h(monitored by TLC and LC-MS). It was then cooled down to rt andconcentrated in vacuo. Crude product was purified by silica gel columnchromatography using a CombiFlash instrument (product eluted at 20% MeOHin EtOAc). Product was isolated as a brown solid (40% yield). ¹H NMR(500 MHz, DMSO-d₆) δ 8.70 (d, J=9.7 Hz, —NH), 7.97 (d, J=6.0 Hz, 1H),7.45 (d, J=6.0 Hz, 1H), 5.70 (td, J=23.6, 9.7 Hz, 1H), 4.14-4.02 (m,8H), 2.50 (s, 3H), 1.22-1.12 (m, 12H). ³¹P NMR (203 MHz, DMSO-d₆) δ16.77 (s). ¹³C NMR (126 MHz, DMSO-d6) δ 167.3, 165.4, 155.12 (t, J=4.1Hz), 121.1, 120.1, 113.6, 62.9-62.7 (m), 44.4 (t, J=147.3 Hz), 16.2-16.1(m), 13.5. MS [ESI⁺] m/z: 484.1 [M+H]⁺.

General Protocol for the Liebeskind-Srogl Cross-Coupling Reaction Shownin Schemes 1 and 2:

The procedure was based on literature with slight modifications (Barbay,J. K. et al. WO 2010/045006; Liebeskind, L. S.; Srogl, J. Org. Lett.2002, 4, 979-981). Therefore tetraethyl(((2-(methylthio)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)(intermediate 4, 882 mg, 1.8 mmol), aryl boronic acid (4.6 mmol; Note:boronic acids were obtained commercially or prepared using establishedmethods), CuTC (1.04 g, 5.5 mmol) and Pd(dppf)Cl₂CH₂Cl₂ (149 mg, 0.18mmol) were charged into an oven-dried round bottom flask. The flask wasevacuated and purged with Ar, followed by addition of dry dioxane (10.0mL). The flask was sealed and heated at 50° C. for 4-16 h (under Arballoon; monitored by TLC and LC-MS). The reaction mixture was cooled toRT, diluted with EtOAc, and filtered through celite. The filtrate wascollected and washed with 10% aqueous NH₄OH (thrice), followed by brine.The combined organic extracts were dried over Na₂SO₄ and concentrated invacuo. Crude product was purified by silica gel column chromatographywith a gradient from 25% EtOAc in hexanes to 100% EtOAc and then to 20%MeOH in EtOAc. Product typically elutes between 10%-20% MeOH in EtOAc.Typical isolated yield was ˜80-85%.

Tetraethyl(((2-(3-nitrophenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)(5a, R^(a)=3-nitrophenyl, R=H) (Scheme 6)

Tetraethyl(((2-(methylthio)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)(4,R^(a)=SMe, R=H; 1.0 eq), 3-nitrophenylboronic acid (2.5 eq), CuTC (3.0eq) and Pd(dppf)Cl₂.CH₂Cl₂ (0.10 eq) were charged into an oven-driedround bottom flask. The flask was evacuated and purged with Ar, followedby addition of dry dioxane (5.5 mL per 1.0 mmol of 4). The flask wassealed and heated at 50° C. for 4 h (under Ar balloon). The reactionmixture was cooled to rt, diluted with EtOAc, and filtered throughcelite. The filtrate was collected and washed with 10% aqueous NH₄OH(thrice), followed by brine. The combined organic extracts were driedover Na₂SO₄ and concentrated in vacuo. Crude product was purified bysilica gel column chromatography (product eluted at 15% MeOH in EtOAc).Product was isolated as light brown solid (80% yield). ¹H NMR (500 MHz,DMSO-d₆) δ 9.12 (t, J=1.9 Hz, 1H), 8.85 (d, J=9.6 Hz, —NH), 8.80 (d,J=7.9 Hz, 1H), 8.35 (ddd, J=8.2, 2.4, 0.9 Hz, 1H), 8.14 (d, J=6.0 Hz,1H), 7.84 (t, J=8.0 Hz, 1H), 7.72 (d, J=6.0 Hz, 1H), 5.99 (td, J=23.5,9.6 Hz, 1H), 4.19-4.07 (m, 8H), 1.22-1.11 (m, 12H). ³¹P NMR (203 MHz,DMSO-d₆) δ 16.9. ¹³C NMR (126 MHz, DMSO-d6) δ 167.8, 156.5 (t, J=4.0Hz), 156.4, 148.7, 139.6, 134.1, 130.8, 125.3, 124.9, 122.4, 120.8,116.4, 63.4-63.2 (m), 45.0 (t, J=147.2 Hz), 16.7-16.6 (m). MS [ESI⁺]m/z: 559.1 [M+H]⁺.

General Protocol for Amide Bond Formation Using Either Method A orMethod B:

Method A: To a stirring solution of 5d in Scheme 6, for example,tetraethyl(((2-(3-aminophenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)(0.15 mmol) in dry DCM (1.5 mL) at 0° C. was added dry Et₃N (0.45 mmol).The acid chloride (Ar—COCl) (0.18 mmol) was then added dropwise. Thesolution was stirred and allowed to warm to RT (reaction progress wasmonitored by TLC and/or LC-MS). Once complete (typically, after ˜1 h),the reaction was poured into saturated aqueous NaHCO₃ solution andextracted with EtOAc (twice), washed with brine, dried over Na₂SO₄ andconcentrated under vacuum. Crude product was purified by silica gelcolumn chromatography with a gradient from 25% EtOAc in hexanes to 100%EtOAc and then to 20% MeOH in EtOAc. Product typically elutes between10%-20% MeOH in EtOAc. Isolated yield was typically 75% to quantitative.Alternatively, aryl carboxylic acid (instead of an acid chloride) wasused in the reaction using HATU or HBTU as the coupling reagentfollowing a similar method described for Method B.

Method B: To the mixture of 5b, in Scheme 6, for example,3-(4-((bis(diethoxyphosphoryl)methyl)amino)thieno[2,3-d]pyrimidin-2-yl)benzoicacid, (0.09 mmol) and amine (Ar—NH₂) (0.1 mmol) in dry DMF (2.0 mL) wasadded DIPEA (0.18 mmol) followed by HBTU (0.1 mmol). The solution wasstirred at RT until completion (typically after ˜1-2 h). Brine was addedto the reaction mixture and was extracted with EtOAc. The organic phasewas washed with sat. NH₄Cl solution, brine, dried over Na₂SO, andconcentrated in vacuo. Crude product was purified by silica-gel columnchromatography as described for Method A, above. Isolated yield wastypically 60-80%.

3-(4-((bis(diethoxyphosphoryl)methyl)amino)thieno[2,3-d]pyrimidin-2-yl)benzoicacid (5b, Scheme 2)

Step 1: Intermediate 4 was reacted with(3-((benzyloxy)carbonyl)phenyl)boronic acid using the general protocolfor the Liebesking-Srogl cross coupling reaction described above toobtain intermediates such as the benzyl3-(4-((bis(diethoxyphosphoryl)methyl)amino)thieno[2,3-d]pyrimidin-2-yl)benzoateintermediate, which was isolated as a yellow solid. ¹H NMR (500 MHz,CD₃OD) δ9.09 (s, 1H), 8.66 (d, J=7.8 Hz, 1H), 8.14 (dd, J=7.7, 1.1 Hz,1H), 7.72 (d, J=6.0 Hz, 1H), 7.64-7.58 (m, 1H), 7.55 (dd, J=6.0, 1.5 Hz,1H), 7.49 (d, J=7.9 Hz, 2H), 7.41 (t, J=7.5 Hz, 2H), 7.36-7.32 (m, 1H),6.19 (t, J=23.5 Hz, 1H), 5.42 (s, 2H), 4.24-4.18 (m, 8H), 1.27-1.21 (m,12H). ³¹P NMR (203 MHz, CD₃OD) δ 17.17 (s). ¹³C NMR (126 MHz, CD₃OD) δ169.5, 167.5, 159.4, 157.4 (t, J=3.9 Hz), 139.7, 137.6, 133.6, 132.2,131.8, 130.1, 129.9, 129.7, 129.3, 129.3, 125.3, 119.8, 117.1, 67.9,65.2-65.1 (m), 45.5 (t, J=150.0 Hz), 16.7-16.6 (m). MS [ESI⁻] m/z: 648.3[M−H]⁻.

Step 2: Synthesis of3-(4-((bis(diethoxyphosphoryl)methyl)amino)thieno[2,3-d]pyrimidin-2-yl)benzoicacid (5b, Scheme 2)

A solution of the above product, benzyl3-(4-((bis(diethoxyphosphoryl)methyl)amino)thieno[2,3-d]pyrimidin-2-yl)benzoate(271 mg, 0.42 mmol), in neat TFA (4.6 mL) was stirred at 80° C. for 14 h(monitored by TLC). TFA was then removed by evaporation in vacuo and theresidue was dissolved in DCM and then evaporated under reduced pressure(done at least twice). Crude product was purified by silica gel columnchromatography using a gradient of 50% EtOAc in hexanes to 100% EtOAcand then to 15% MeOH in EtOAc. Product was isolated as a light brownsolid (quantitative yield). ¹H NMR (400 MHz, CDCl₃ with ˜0.1% CD₃OD) δ9.20 (s, 1H), 8.70 (d, J=7.9 Hz, 1H), 8.19 (d, J=7.7 Hz, 1H), 7.70 (d,J=6.0 Hz, 1H), 7.59 (t, J=7.8 Hz, 1H), 7.40 (d, J=6.0 Hz, 1H), 6.08 (t,J=22.2 Hz, 1H), 4.29-4.18 (m, 8H), 1.29 (t, J=7.0 Hz, 6H), 1.20 (t,J=7.0 Hz, 6H). ³¹P NMR (203 MHz, CDCl₃) δ 17.50 (s). ¹³C NMR (126 MHz,CDCl₃ with ˜0.1% CD₃OD) δ 169.2, 168.3, 158.4, 155.8, 138.4, 132.7,131.5, 129.8, 128.6, 123.8, 118.8, 115.9, 115.8, 64.2 (br), 44.1 (t,J=147.0 Hz, 1H), 16.2 (br). MS [ESI⁺] m/z: 558.1 [M+H]⁺.

Tetraethyl(((2-(3-aminophenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)(5d; Q=CH, q=1; R=H) (Scheme 6)

A pressure vessel was charged with tetraethyl(((2-(3-nitrophenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)(5a, R^(b)=3-NO₂, Q=CH, q=1, R=H; 1.0 eq) and EtOH (0.1 M). SnCl₂.2H₂O(5.0 eq) was then added and the mixture was stirred at 80° C. for 2-3 h,cooled to rt and the mixture was slowly added to sat. NaHCO₃ solution(10.0 mL) and then extracted with EtOAc (thrice). The combined organicphase was washed with brine, dried over MgSO₄, filtered, andconcentrated in vacuo. Product was isolated as a yellow solid (80%yield) and used in the next step without further purification. ¹H NMR(500 MHz, DMSO-d₆) δ 8.52 (d, J=9.7 Hz, 1H), 8.06 (d, J=6.0 Hz, 1H),7.64-7.62 (m, 1H), 7.58 (d, J=6.0 Hz, 1H), 7.53 (d, J=7.7 Hz, 1H), 7.14(t, J=7.8 Hz, 1H), 6.67 (dd, J=7.9, 1.5 Hz, 1H), 6.01 (td, J=23.5, 9.7Hz, 1H), 5.22 (s, 2H), 4.15-4.05 (m, 8H), 1.18-1.10 (m, 12H). ³¹P NMR(203 MHz, DMSO-d6) δ 17.18 (s). MS [ESI⁺] m/z: 529.1 [M+H]⁺.

General procedures for the preparation of the tetraethyl bisphosphonateester intermediates from the C-4 amine, followed by deprotection of theesters to give the phosphonic acids were carried out as previouslyreported [as examples refer to (a) Leung, et al. J. Med. Chem. 2013, 56,7939-7950. (b) Leung, et al Bioorg. Med. Chem. 2013, 21, 2229-2240.]

Comparative Examples(((2-(furan-2-yl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonicacid) (C-1) (Table 1)

Prepared using the method shown in Scheme 1. Isolated as a light brownsolid (73% yield). ¹H NMR (500 MHz, D₂O) δ 7.71 (s, 1H), 7.45 (d, J=5.9Hz, 1H), 7.40 (d, J=5.9 Hz, 1H), 7.35 (d, J=3.2 Hz, 1H), 6.63 (br, 1H),5.22 (t, J=20.3 Hz, 1H). ³¹P NMR (203 MHz, D₂O): δ 12.9. ¹³C NMR (125MHz, D₂O): δ 163.4, 156.5, 151.4, 150.1, 145.6, 123.5, 118.8, 115.6,114.0, 112.5, 48.8 (t, J=130.5 Hz). MS (ESI⁺) m/z: 392.1 [M+H]⁺.

(((2-(pyridin-4-yl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonicacid) (C-2) (Table 1)

Prepared using this method shown in Scheme 1. Isolated as a yellow solid(76% yield). ¹H NMR (400 MHz, D₂O): δ 8.60 (br, 2H), 8.24 (br, 2H), 7.58(d, J=6.0 Hz, 1H), 7.52 (d, J=5.9 Hz, 1H), 5.26 (t, J=19.4 Hz, 1H). ³¹PNMR (162 MHz, D₂O): δ 13.7. ¹³C NMR (100 MHz, D₂O): δ 165.4, 157.4,156.9, 148.5, 146.5, 124.4, 122.8, 118.8, 116.5, 48.8 (t, J=124.0 Hz).MS (ESI⁺) m/z: 403.1 [M+H]⁺.

Representative Compounds of Formula I

(((2-([1,1′-biphenyl]-3-yl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonicacid) (I-1) (Table 2)

Prepared using the method shown in Scheme 6.

Step 1: Tetraethyl(((2-(3-bromophenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)(5c, R^(b)=Br, R=H; 1 eq), [1,1′-biphenyl]-3-ylboronic acid (1.3 eq),Pd(PPh₃)₄ (10 mol %), and KF (2.5 eq) were placed in a microwave reactorvial and the mixture was purged with Argon (Ar). The mixture was addedwith MeOH (2.5 mL per 0.10 mmol of 5c) and purged again with Ar. Thereaction mixture was heated via a microwave (120° C.) for 20 min. Thereaction mixture was cooled down, filtered through celite, andconcentrated to dryness under vacuum. Crude product was purified bysilica gel column chromatography using a solvent gradient of 5% to 100%EtOAc in hexanes and then from 0% to 20% MeOH in EtOAc. Tetraethyl(((2-([1,1′-biphenyl]-3-yl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)(R²=[1,1′-biphenyl]-3-yl; R=H) was isolated as a light yellow solid (82%yield). ¹H NMR (500 MHz, CDCl₃) δ 8.74 (s, 1H), 8.45 (d, J=7.8 Hz, 1H),7.73-7.69 (m, 3H), 7.57 (t, J=7.7 Hz, 1H), 7.48 (t, J=7.6 Hz, 2H),7.40-7.36 (m, 3H), 6.02-5.91 (m, 2H; —NH and α-CH to thebisphosphonate), 4.30-4.13 (m, 8H), 1.29-1.22 (m, 12H). ³¹P NMR (203MHz, CDCl₃) δ 16.76.

Step 2: Deprotection of tetraethyl bisphosphonate ester. Final product(I-1) was afforded as a light brown solid (75% yield). ¹H NMR (500 MHz,D₂O) δ 8.52 (s, 1H), 8.31 (d, J=7.7 Hz, 1H), 7.88-7.76 (m, 3H), 7.66 (t,J=7.8 Hz, 1H), 7.60 (d, J=5.9 Hz, 1H), 7.55 (t, J=7.7 Hz, 2H), 7.48 (d,J=6.0 Hz, 1H), 7.44 (d, J=7.4 Hz, 1H), 5.19 (t, J=17.7 Hz, 1H). ³¹P NMR(203 MHz, D₂O): δ 13.9. ¹³C NMR (125 MHz, D₂O): δ 165.7 160.4, 157.0,141.0, 140.3, 138.3, 129.4, 129.1, 129.0, 127.8, 127.5, 127.1, 126.4,123.2, 118.8, 115.7. Cα observed by HSQC. HSQC (¹H-¹³C): ¹H δ 5.19correlates with ¹³C δ 49.0. MS (ESI⁺) m/z: 478.2 [M+H]⁺.

(((2-(3-(phenylsulfonamido)phenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonicacid) (I-2) (Table 2)

Prepared using the method shown in Scheme 6.

Step 1: To the solution of tetraethyl(((2-(3-aminophenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)(5d, Q=CH, q=1, 3-amino; R=H; 1.0 eq) in dry DCM (1.3 mL per 0.1 mmol5d) at 0° C. was added pyridine (7.5 eq). Benzenesulfonyl chloride (1.2eq) was then added and the reaction was allowed to warm to rt (reactionprogress was monitored by TLC). Once complete (typically, after ˜1-2 h),the reaction was poured into sat. NaHCO₃ solution, extracted with EtOAc(thrice), washed with 1M HCl, and the combined organic phase was driedover MgSO₄, and then concentrated in vacuo. Crude product was purifiedby silica gel column chromatography (product eluted at 5% MeOH inEtOAc). Tetraethyl(((2-(3-(phenylsulfonamido)phenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)was isolated as a cream-color solid (73%). ¹H NMR (500 MHz, DMSO-d₆) δ10.61 (s, —NH), 8.58 (d, J=9.7 Hz, —NH), 8.22 (d, J=8.8 Hz, 2H), 8.05(d, J=6.0 Hz, 1H), 7.84-7.82 (m, 2H), 7.63-7.55 (m, 4H), 7.24 (d, J=8.8Hz, 2H), 5.94 (td, J=23.3, 9.4 Hz, 1H), 4.13-4.04 (m, 8H), 1.16-1.08 (m,12H). ³¹P NMR (203 MHz, DMSO-d₆) δ 17.12 (s).

Step 2: Deprotection of tetraethyl bisphosphonate ester. Final product(I-2) was afforded as a cream solid (88% yield). ¹H NMR (500 MHz, D₂O) δ8.20 (d, J=8.3 Hz, 2H), 7.87 (d, J=7.6 Hz, 2H), 7.68-7.63 (m, 1H),7.59-7.56 (m, 3H), 7.45 (d, J=5.8 Hz, 1H), 7.22 (d, J=8.4 Hz, 2H), 5.15(t, J=17.0 Hz, 1H). ³¹P NMR (203 MHz, D₂O) δ 13.87 (s). ¹³C NMR (101MHz, D₂O) δ 165.7, 159.7, 156.9, 140.4, 138.2, 133.4 (two carbons),129.4 (two carbons), 129.3, 129.2, 126.8, 122.8, 121.3, 118.8, 115.5.C-α to the bisphosphonate was observed by HSQC. HSQC (¹H-¹³C): ¹H at δ5.15 correlates to ¹³C-α at δ 47.0. HRMS [ESI⁺] calculated forC₁₉H₁₆N₄Na₃O₈P₂S₂ m/z, 622.95724; found 622.95769 [M+3 Na]⁺.

(((2-(3-(thiophene-2-carboxamido)phenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonicacid) (I-3) (Table 2)

Prepared using the method shown in Scheme 6.

Step 1: To the mixture of tetraethyl(((2-(3-aminophenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)(5d, Z=CH, n=1, 3-amino; RH; 1.0 eq) and 2-thiophenecarboxylic acid (1.1eq) in dry DMF (2.0 mL per 0.1 mmol 6) was added DIPEA (2.0 eq) followedby HBTU (1.1 eq) under an Ar balloon. The solution was stirred at rt for4 h (monitored by TLC and LC-MS). The reaction was then added with brine(10 mL) and was extracted with EtOAc (20.0 mL; twice). The combinedorganic phases were washed with sat. NH₄Cl solution (10 mL), brine,dried over Na₂SO₄ and concentrated in vacuo. Crude product was purifiedby silica-gel column chromatography with a gradient from 25% EtOAc inhexanes to 100% EtOAc and then to 20% MeOH in EtOAc (product eluted at10% MeOH in EtOAc). Tetraethyl(((2-(3-(thiophene-2-carboxamido)phenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)was isolated as a yellow solid (77%). ¹H NMR (500 MHz, DMSO-d₆) δ 10.41(s, —NH), 8.80 (t, J=1.8 Hz, 1H), 8.66 (d, J=9.7 Hz, —NH), 8.12 (d,J=7.9 Hz, 1H), 8.10-8.09 (m, 2H), 7.89-7.87 (m, 2H), 7.64 (d, J=6.0 Hz,1H), 7.51 (t, J=7.9 Hz, 1H), 7.25 (dd, J=4.9, 3.8 Hz, 1H), 6.06 (td,J=23.4, 9.7 Hz, 1H), 4.18-4.06 (m, 8H), 1.20-1.10 (m, 12H). ³¹P NMR (203MHz, DMSO-d₆) δ 16.97 (s). MS [ESI⁺] m/z: 639.1 [M+H⁺]⁺.

Step 2: Deprotection of tetraethyl bisphosphonate ester. Final product(I-3) was afforded as yellow solid (71% yield). ¹H NMR (400 MHz, D₂O) δ8.35 (s, 1H), 8.17 (d, J=7.9 Hz, 1H), 7.94 (dd, J=3.7, 0.8 Hz, 1H), 7.86(dd, J=8.0, 1.2 Hz, 1H), 7.80 (dd, J=5.0, 0.9 Hz, 1H), 7.64-7.60 (m,2H), 7.49 (d, J=6.0 Hz, 1H), 7.27 (dd, J=4.9, 3.9 Hz, 1H), 5.14 (t,J=18.8 Hz, 1H). ³¹P NMR (203 MHz, D₂O) δ 13.81 (s). ¹³C NMR (101 MHz,D₂O) δ 165.5, 163.3, 159.8, 156.7, 138.7, 137.6, 137.2, 132.2, 130.4,129.5, 128.4, 125.2, 124.3, 123.0, 121.9, 119.0, 115.9. C-α to thebisphosphonate was observed by HSQC. HSQC (¹H-¹³C): ¹H at δ 5.14correlates to ¹³C-α at δ 49.5. HRMS [ESI⁺] calculated forC₁₈H₁₄N₄Na₃O₇P₂S₂ m/z, 592.94667; found 592.94813 [M+3Na]⁺.

(((2-(3-(cyclohexanecarboxamido)phenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonicacid) (I-4) (Table 2)

Prepared following the protocol described for I-3. Step 1: Theintermediate, tetraethyl(((2-(3-(cyclohexanecarboxamido)phenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)(R^(c)=3-(cyclohexanecarboxamido)phenyl; R=H) was isolated as a yellowsolid (68% yield). ¹H NMR (500 MHz, DMSO-d6) δ 9.97 (s, —NH), 8.68 (t,J=1.8 Hz, 1H), 8.62 (d, J=9.7 Hz, —NH), 8.09 (d, J=6.0 Hz, 1H), 8.03 (d,J=7.9 Hz, 1H), 7.71 (ddd, J=8.1, 2.0, 0.9 Hz, 1H), 7.62 (d, J=6.0 Hz,1H), 7.42 (t, J=7.9 Hz, 1H), 6.04 (td, J=23.4, 9.7 Hz, 1H), 4.18-4.06(m, 8H), 2.37 (tt, J=11.6, 3.4 Hz, 1H), 1.78-1.65 (m, 5H), 1.48-1.20 (m,5H), 1.19-1.09 (m, 12H). ³¹P NMR (203 MHz, DMSO-d6) δ 16.98 (s). MS[ESI⁺] m/z: 639.2 [M+H]⁺.

Step 2: Final product (I-4) was afforded as a yellow solid (81% yield).¹H NMR (500 MHz, D₂O) δ 8.25 (s, 1H), 8.14 (d, J=7.9 Hz, 1H), 7.82 (d,J=8.1 Hz, 1H), 7.64-7.59 (m, 2H), 7.50 (d, J=5.9 Hz, 1H), 5.11 (t,J=19.1 Hz, 1H), 2.49 (tt, J=11.8, 3.3 Hz, 1H), 1.99-1.73 (m, 5H),1.56-1.27 (m, 5H). ³¹P NMR (203 MHz, D₂O) δ 13.77 (s). ¹³C NMR (101 MHz,D₂O) δ 179.1, 165.6, 159.9, 157.0, 138.6, 137.5, 129.5, 124.9, 123.8,123.1, 121.4, 118.9, 115.8, 45.8, 29.1, 25.3, 25.2. C-α to thebisphosphonate was observed by HSQC. HSQC (¹H-¹³C): ¹H at 5 5.11correlates to ¹³C-α at δ 49.0. HRMS [ESI⁺] calculated forC₂₀H₂₂N₄Na₃O₇P₂S m/z, 593.03720; found 593.03804 [M+3 Na]⁺.

(((2-(3-(4-methoxybenzamido)phenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonicacid) (I-5) (Table 2)

Prepared using the method shown in Scheme 6.

Step 1: To a stirring solution of tetraethyl(((2-(3-aminophenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)(5d, Q=CH, q=1, 3-amino; R H; 1.0 eq) in dry DCM (6.5 mL per 1 mmol of5d) at 0° C. was added dry Et₃N (3.0 eq). Para-methoxybenzoyl chloride(1.2 eq) was then added dropwise under Ar balloon. The solution wasstirred and allowed to warm to RT (reaction progress was monitored byTLC or LC-MS). Once complete (typically, after ˜1 h), the reactionmixture was poured into sat. NaHCO₃ solution and extracted with EtOAc(twice), washed with brine, dried over Na₂SO₄ and concentrated undervacuum. Crude product was purified by silica gel column chromatographywith a gradient from 25% EtOAc in hexanes to 100% EtOAc and then to 20%MeOH in EtOAc (product eluted at 15% MeOH in EtOAc).

Tetraethyl(((2-(3-(4-methoxybenzamido)phenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)was isolated as a yellow solid (quantitative yield). ¹H NMR (500 MHz,DMSO-d₆) δ 10.27 (s, —NH), 8.84 (t, J=1.7 Hz, 1H), 8.65 (d, J=9.7 Hz,—NH), 8.11-8.09 (m, 2H), 8.02 (d, J=8.8 Hz, 2H), 7.90 (dd, J=6.9, 1.2Hz, 1H), 7.64 (d, J=6.0 Hz, 1H), 7.49 (t, J=7.9 Hz, 1H), 7.08 (d, J=8.9Hz, 2H), 6.06 (td, J=23.4, 9.7 Hz, 1H), 4.18-4.07 (m, 8H), 3.85 (s, 3H),1.19-1.10 (m, 12H). ³¹P NMR (203 MHz, DMSO-d6) δ 17.01 (s). MS [ESI⁺]m/z: 663.2 [M+H]⁺.

Step 2: Deprotection of tetraethyl bisphosphonate ester. Final product(I-5) was afforded as a beige solid (81%). ¹H NMR (400 MHz, D₂O) δ 8.36(s, 1H), 8.19 (d, J=7.8 Hz, 1H), 7.98 (d, J=8.8 Hz, 2H), 7.91 (d, J=8.1Hz, 1H), 7.68-7.60 (m, 2H), 7.50 (d, J=6.0 Hz, 1H), 7.17 (d, J=8.9 Hz,2H), 5.11 (t, J=19.0 Hz, 1H), 3.94 (s, 3H). ³¹P NMR (162 MHz, D₂O) δ13.78 (s). ¹³C NMR (126 MHz, D₂O) δ 169.1, 165.0, 162.2, 160.0, 156.8,138.7, 137.6, 129.6, 129.5, 126.3, 125.1, 124.4, 122.6, 122.0, 119.2,116.0, 114.1, 55.5. C-α to the bisphosphonate was observed by HSQC. HSQC(¹H-¹³C): ¹H at δ 5.11 correlates to ¹³C-α at δ 50.0. HRMS [ESI⁺]calculated for C₂₁H₁₈N₄Na₃O₈P₂S m/z, 617.00082; found 617.00181 [M+3Na]⁺.

(((2-(3-(phenylcarbamoyl)phenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonicacid) (I-34) (Table 2)

Prepared using the method shown in Scheme 2.

Step 1: A solution of 5d [tetraethyl(((2-(3-aminophenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)]was reacted with the 4-fluorobenzoic acid in dry DMF in the presence ofDIPEA (2 eq) and HBTU (1.1 eq) under argon balloon at RT. The reactionwas followed by TLC, upon completion of the reaction, brine (10 ml) wasadded and the mixture is extracted with ethyl acetate. The organic phasewas washed with saturated ammonium chloride (10 ml), brine, dried oversodium sulfate and concentrated in vacuo. Crude product was purified bysilica-gel column chromatography using a Combiflash instrument. Producteluted at 15% methanol in ethyl acetate and was obtained as a yellowsolid (48 mg, 65%). ¹H NMR (500 MHz, CDCl₃) δ 8.39 (s, 1H), 8.28 (d,J=7.9 Hz, 1H), 8.14 (d, J=7.6 Hz, 1H), 8.03-7.90 (m, 3H), 7.52 (t, J=8.0Hz, 1H), 7.37 (d, J=6.0 Hz, 1H), 7.21 (t, J=8.5 Hz, 2H), 5.97 (d, J=9.9Hz, 1H), 5.78 (s, 1H), 4.35-4.08 (m, 8H), 1.29-1.19 (m, 12H). ³¹P NMR(203 MHz, CDCl₃) δ 16.84.

After deprotection of the tetraethyl esters, the final compound I-34 wasisolated as a beige powder (26.1 mg, 63%). ¹H NMR (400 MHz, D₂O) δ 8.37(s, 1H), 8.20 (d, J=7.9 Hz, 1H), 8.05-7.98 (m, 2H), 7.93 (d, J=8.1 Hz,1H), 7.70-7.60 (m, 2H), 7.51 (d, J=6.0 Hz, 1H), 7.39-7.29 (m, 2H), 5.17(t, J=19.0 Hz, 1H). ³¹P NMR (162 MHz, D₂O) δ 13.93 (s). ¹³C NMR (126MHz, D₂O) δ 168.5, 165.8, 165.4, 163.8, 159.7, 156.9, 138.5, 137.4,130.1, 130.0, 129.4, 125.1, 124.1, 122.9, 121.7, 118.9, 115.8, 115.7,115.5, 49.5 (t, J=122.7 Hz). HRMS [ESI⁻] calculated for C₂₀H₁₅FN₄NaO₇P₂Sm/z [M-2H+Na]⁻ 559.0024; found 559.0010 [M-2H+Na]⁻.

(((2-(3-(phenylcarbamoyl)phenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonicacid) (I-6) (Table 2)

Tetraethyl(((2-(3-(phenylcarbamoyl)phenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)was isolated as light yellow solid (84% yield). ¹H NMR (400 MHz,DMSO-d₆) δ 10.47 (s, —NH), 8.95 (s, 1H), 8.75 (d, J=9.6 Hz, —NH), 8.57(d, J=7.9 Hz, 1H), 8.10 (d, J=5.7 Hz, 1H), 8.07 (d, J=7.9 Hz, 1H), 7.82(d, J=7.7 Hz, 2H), 7.71-7.66 (m, 2H), 7.39-7.35 (m, 2H), 7.12 (t, J=7.4Hz, 1H), 6.03 (br, 1H), 4.18-4.00 (m, 8H), 1.19-1.08 (m, 12H). ³¹P NMR(162 MHz, DMSO-d₆) δ 17.05 (s). ¹³C NMR (126 MHz, DMSO-d₆) δ 167.6,165.5, 157.5, 156.0 (t, J=3.7 Hz), 139.2, 137.7, 135.5, 130.4, 129.4,128.7, 128.6, 127.1, 123.8, 123.7, 120.3, 120.2, 115.5, 62.9-62.7 (m),16.2-16.1 (m). C-α to the bisphosphonate was observed by HSQC. HSQC(¹H-¹³C): ¹H at δ 6.03 correlates to ¹³C-α at δ 45.1. MS [ESI⁺] m/z:633.2 [M+H]⁺.

Step 2: Deprotection of tetraethyl bisphosphonate ester. Final product(I-6) was isolated as off-white solid (67%). ¹H NMR (400 MHz, D₂O) δ8.81 (s, 1H), 8.58 (d, J=7.9 Hz, 1H), 8.06 (d, J=7.8 Hz, 1H), 7.76 (t,J=7.8 Hz, 1H), 7.66-7.63 (m, 3H), 7.56-7.52 (m, 3H), 7.36 (t, J=7.4 Hz,1H), 5.20 (t, J=19.0 Hz, 1H). ³¹P NMR (203 MHz, D₂O) δ 13.91 (s). ¹³CNMR (126 MHz, D₂O) δ 169.4, 165.5, 159.7, 157.0, 138.3, 136.9, 134.4,132.0, 129.4, 129.3, 129.2, 126.9, 126.0, 123.2, 123.0, 119.0, 115.9,49.3. HRMS [ESI⁺] calculated for C₂₀H₁₆N₄Na₃O₇P₂S m/z, 586.9903; found586.9903 [M+3 Na]⁺.

(((2-(3-(3-(4-fluorophenyl)ureido)phenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonicacid) (I-7) (Table 2)

Prepared using the method shown in Scheme 6.

Step 1: To the mixture of triphosgene (1.0 eq) in dry toluene (0.05 M)was added 4-fluoroaniline (1.5 eq) at RT under Ar. Et₃N (1.1 eq) wasthen added dropwise and the reaction mixture was refluxed for 4 h. Thesolvent was removed in vacua and the residue was re-dissolved in dry DCM(1.0-2.0 mL). This was then added to the solution of tetraethyl(((2-(3-aminophenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)(5d, Q=CH, q=1, 3-amino; R=H; 0.80 eq) and Et₃N (1.2 eq) in dry DCM (1.0mL per 0.1 mmol 5d) in an ice-bath. The resulting mixture was stirred atrt until completion (typically, after 12 h). The solvent was thenremoved in vacuo and the residue was dissolved in EtOAc, washed withsaturated NH₄Cl, brine, dried over Na₂SO₄, and the organic extract wascollected and concentrated in vacuo. Crude product was purified bysilica-gel chromatography using a Combiflash instrument (product elutedat ˜10% MeOH in EtOAc). Tetraethyl(((2-(3-(3-(4-fluorophenyl)ureido)phenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate))was isolated as a yellow solid (71% yield). ¹H NMR (500 MHz, CDCl₃) δ8.12-8.01 (m, 4H), 8.08 (s, 1H), 7.79 (s, 1H), 7.42-7.37 (m, 3H), 7.28(d, J=7.8 Hz, 1H), 7.21 (d, J=5.6 Hz, 1H), 6.91 (t, J=8.7 Hz, 2H), 6.74(br_s, 1H), 6.20 (t, J=25.8 Hz, 1H), 4.32-4.20 (m, 8H), 1.29-1.24 (m,12H). ³¹P NMR (203 MHz, CDCl₃) δ 17.14 (s). MS [ESI⁺] m/z: 666.2 [M+H]⁺.

Step 2: Deprotection of tetraethyl bisphosphonate ester. Final product(I-7) was afforded as a yellow solid (74% yield). ¹H NMR (400 MHz,DMSO-d₆) δ 8.90 (br_s, 1H), 8.73 (br_s, 1H), 8.34 (s, 1H), 8.05 (d,J=7.8 Hz, 1H), 7.89 (br, 1H), 7.73 (d, J=7.5 Hz, 2H), 7.57 (d, J=5.6 Hz,1H), 7.50-7.47 (m, 2H), 7.41 (t, J=7.9 Hz, 1H), 7.12 (t, J=8.7 Hz, 2H),5.62 (br_, 1H). ³¹P NMR (162 MHz, DMSO-d₆) δ 13.98 (s). MS [ESI⁺] m/z:554.0 [M+H]⁺.

(((2-(3-(4-methylbenzamido)phenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonicacid) (I-18) (Table 2)

Step 1: Tetraethyl(((2-(3-(4-methylbenzamido)phenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)was isolated as a yellow solid. ¹H NMR (500 MHz, DMSO-d₆) δ 10.34 (s,—NH), 8.85 (t, J=1.8 Hz, 1H), 8.65 (d, J=9.7 Hz, —NH), 8.12-8.09 (m,2H), 7.93 (d, J=8.2 Hz, 2H), 7.91 (ddd, J=8.1, 2.1, 1.0 Hz, 1H), 7.64(d, J=6.0 Hz, 1H), 7.50 (t, J=7.9 Hz, 1H), 7.35 (d, J=7.9 Hz, 2H), 6.06(td, J=23.4, 9.7 Hz, 1H), 4.18-4.05 (m, 8H), 2.40 (s, 3H), 1.19-1.10 (m,12H). ³¹P NMR (203 MHz, DMSO-d₆) δ 17.00 (s). MS [ESI⁺] m/z: 647.2[M+H]⁺.

Step 2: Compound I-18 was isolated as a light yellow solid. ¹H NMR (500MHz, D₂O) δ 8.38 (s, 1H), 8.20 (d, J=7.6 Hz, 1H), 7.93 (d, J=7.1 Hz,1H), 7.89 (d, J=7.9 Hz, 2H), 7.68-7.63 (m, 2H), 7.51 (d, J=5.9 Hz, 1H),7.46 (d, J=7.9 Hz, 2H), 5.15 (t, J=18.9 Hz, 1H), 2.47 (s, 3H). ³¹P NMR(203 MHz, D₂O) δ 13.82 (s). ¹³C NMR (101 MHz, D₂O) δ 169.3, 165.6,159.5, 156.8, 143.5, 138.3, 137.5, 130.7, 129.4, 129.3, 127.5, 125.0,124.1, 123.3, 121.6, 118.8, 115.8, 48.9 (t, J=124.8 Hz), 20.6. HRMS[ESI⁺] calculated for C₂₁H₁₈N₄Na₃O₇P₂S m/z 601.00590; found 601.00773[M+3 Na]⁺.

(((2-(3-((4-methoxyphenyl)carbamoyl)phenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonicacid) (I-36) (Table 2)

Step 1: Tetraethyl(((2-(3-((4-methoxyphenyl)carbamoyl)phenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)was isolated as a light brown solid. ¹H NMR (500 MHz, CDCl₃ with ˜0.1%CD₃OD) δ 9.01 (s, 1H), 8.67 (br_s, 1H), 8.59 (d, J=7.8 Hz, 1H), 8.11 (d,J=7.6 Hz, 1H), 7.68 (d, J=8.8 Hz, 2H), 7.61 (t, J=7.7 Hz, 1H), 7.45 (d,J=5.3 Hz, 1H), 7.37 (d, J=6.0 Hz, 1H), 6.92 (d, J=9.0 Hz, 2H), 5.86 (t,J=22.3 Hz, 1H), 4.27-4.10 (m, 8H), 3.82 (s, 3H), 1.26 (t, J=7.1 Hz, 6H),1.21 (t, J=7.1 Hz, 6H). ³¹P NMR (203 MHz, CDCl₃) δ 17.10 (s). MS [ESI⁺]m/z: 663.4 [M+H]⁺.

Step 2: Compound I-36 was isolated as off-white solid. ¹H NMR (500 MHz,D₂O) δ 8.74 (s, 1H), 8.54 (d, J=7.8 Hz, 1H), 8.00 (d, J=7.7 Hz, 1H),7.72 (t, J=7.8 Hz, 1H), 7.62 (d, J=5.9 Hz, 1H), 7.51-7.49 (m, 3H), 7.06(d, J=8.9 Hz, 2H), 5.18 (t, J=18.9 Hz, 1H), 3.86 (s, 3H). ¹³C NMR (126MHz, D₂O) δ 169.3, 165.5, 159.7, 157.0, 156.7, 138.3, 134.3, 132.0,130.1, 129.3, 129.3, 126.8, 124.8, 123.2, 118.9, 115.9, 114.4, 55.5.HSQC (¹H-¹³C): ¹H at δ 5.18 correlates to ¹³C-α at δ 49.3. ³¹P NMR (203MHz, D₂O) δ 13.91 (s). HRMS [ESI⁺] calculated for C₂₁H₁₈N₄Na₃O₈P₂S m/z,617.00082; found 617.00090 [M+3Na]⁺.

(((2-(3-((3-fluoro-4-methoxyphenyl)carbamoyl)phenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonicacid) (I-37) (Table 2)

Step 1: Tetraethyl(((2-(3-((3-fluoro-4-methoxyphenyl)carbamoyl)phenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)was isolated as a yellow solid. ¹H NMR (500 MHz, CDCl₃) δ 9.28 (s, 1H),9.10 (br_s, 1H), 8.62 (d, J=7.8 Hz, 1H), 8.14 (d, J=7.7 Hz, 1H), 7.70(dd, J=13.1, 2.3 Hz, 1H), 7.66 (d, J=8.7 Hz, 1H), 7.59 (t, J=7.7 Hz,1H), 7.42 (d, J=5.8 Hz, 1H), 7.27 (d, J=6.0 Hz, 1H), 6.94 (t, J=9.1 Hz,1H), 6.77 (br_s, 1H), 5.63 (td, J=22.9, 7.7 Hz, 1H), 4.24-4.11 (m, 8H),1.24-1.20 (m, 12H). ³¹P NMR (203 MHz, CDCl₃) δ 17.27 (s). ¹³C NMR (126MHz, CDCl₃) δ 168.4, 165.3, 158.5, 155.8 (t, J=3.3 Hz), 152.2 (d,J=244.1 Hz), 144.3 (d, J=10.9 Hz), 138.0, 134.7, 132.6 (d, J=9.4 Hz),131.1, 130.2, 129.1, 126.8, 123.9, 117.9, 116.3 (d, J=3.4 Hz), 115.5,113.7 (d, J=2.5 Hz), 109.7 (d, J=22.6 Hz), 63.9-63.7 (m), 56.7, 46.9 (t,J=147.9 Hz), 16.5-16.4 (m). MS [ESI⁺] m/z: 681.3 [M+H]⁺.

Step 2: Compound I-37 was isolated as a light yellow solid. ¹H NMR (500MHz, D₂O) δ 8.77 (s, 1H), 8.55 (d, J=7.8 Hz, 1H), 8.03 (d, J=7.8 Hz,1H), 7.74 (t, J=7.8 Hz, 1H), 7.64 (d, J=5.9 Hz, 1H), 7.53-7.51 (m, 2H),7.34 (d, J=8.5 Hz, 1H), 7.23 (t, J=9.1 Hz, 1H), 5.21 (t, J=18.5 Hz, 1H),3.95 (s, 3H). ³¹P NMR (203 MHz, D₂O) δ 13.90 (s). ¹³C NMR (126 MHz, D₂O)δ 168.8, 165.5, 159.5, 157.0, 151.3 (d, J=242.1 Hz), 144.3 (d, J=10.9Hz), 138.1, 133.9, 132.0, 130.5 (d, J=9.4 Hz), 129.3, 129.2, 126.8,123.2, 118.7, 118.7 (d, J=3.1 Hz). 115.8, 113.9 (d, J=1.9 Hz), 111.0 (d,J=21.8 Hz), 56.3. HSQC (¹H-¹³C): ¹H at δ 5.19 correlates to ¹³C-α at δ49.0. HRMS [ESI⁺] calculated for C₂₁H₁₇O₈N₄FNa₃P₂S m/z, 634.99140; found634.99164 [M+3 Na]⁺.

Synthesis of 2-(methylthio)thieno[3,2-d]pyrimidin-4(3H)-one(intermediate 10 in Scheme 2)

Methyl-3-amino-2-thiophenecarboxylate (1.00 g, 6.36 mmol) was added to4M hydrochloric acid in dioxane (9.5 ml), followed by methyl thiocyanate(465 mg, 6.36 mmol). The resulting suspension was heated to 90° C. in asealed pressure tube for 24 h. The reaction was followed by TLC, uponcompletion the mixture was allowed to cool to RT and the resulting whiteprecipitate was collected by vacuum filtration. The solid was washedwith ethanol followed by hexane. The resulting white solid (1.19 g, 94%)was used in the next step without any further purification.

¹H NMR (500 MHz, DMSO-d₆) δ 8.14 (d, J=5.2 Hz, 1H), 7.31 (d, J=5.2 Hz,1H), 2.54 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 158.2, 157.7, 157.3, 135.1,124.5, 119.1, 12.9.

4-chloro-2-(methylthio)thieno[3,2-d]pyrimidine (intermediate 11 inScheme 2)

Phosphorus oxychloride (17.9 ml, 192 mmol) was added to compound 10(3.80 g, 19.2 mmol) in a reaction flask and heated at 106° C. (reflux)for 12 h. The reaction was followed by TLC. When reaction was completed,the phosphorus oxychloride was removed by distillation and the remainingreaction mixture was cooled down to 0° C., then it was quenched bydropwise addition of saturated aqueous sodium bicarbonate until pH 7 wasobtained. The aqueous layer was extracted with DCM (thrice) and thecombined organic layers were washed with brine and then concentratedunder reduced pressure. The product was obtained as a white powder (3.04g, 73%). ¹H NMR (500 MHz, CDCl₃) δ 7.98 (d, J=5.5 Hz, 1H), 7.45 (d,J=5.5 Hz, 1H), 2.65 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 168.9, 162.5,154.6, 137.5, 126.1, 124.2, 14.7. MS [ESI⁺] m/z: 217.03 and 219.03[M+H]⁺.

2-(methylthio)thieno[3,2-d]pyrimidin-4-amine (intermediate 12 in Scheme2)

Ammonium hydroxide 28% (18.7 ml, 592 mmol) was added to intermediate 11(1.28 g, 5.93 mmol). The reaction was heated to 90° C. for 12 h(overnight). The reaction was followed by TLC. The mixture was thenallowed to cool to RT, filtered, washed with water and air dried. Theproduct was obtained as a light green powder (1.06 g, 91%). ¹H NMR (500MHz, DMSO) δ 8.06 (d, J=5.4 Hz, 1H), 7.48 (s, 2H), 7.26 (d, J=5.3 Hz,1H), 2.46 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 166.7, 160.5, 157.7, 133.7,123.6, 110.8, 13.3. MS [ESI⁺] m/z: 198.05 [M+H]⁺.

Tetraethyl (((2-(methylthio)thieno[3,2-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate) (intermediate 13 in Scheme 2)

A pressure vessel charged with compound 12 (750 mg, 3.79 mmol) intoluene (2.2 ml) was added to diethyl phosphite (3.4 ml, 26.5 mmol) andtriethyl orthoformate (1.1 ml, 6.45 mmol). The mixture was heated at130° C. for 48 h and monitored by TLC and LC-MS. Cooled down andconcentrated in vacuo. Crude product was purified by silica gel columnchromatography. The product elutes at 20% methanol in ethyl acetate.Product was obtained as yellow solid (1.00 g, 55%). ¹H NMR (500 MHz,CDCl₃) δ 7.73 (d, J=5.3 Hz, 1H), 7.33 (d, J=5.3 Hz, 1H), 5.70 (td,J=21.9, 9.8 Hz, 1H), 5.49 (d, J=9.2 Hz, 1H), 4.29-4.12 (m, 8H), 2.58 (s,3H), 1.33-1.22 (m, 12H). ³¹P NMR (203 MHz, CDCl₃) δ 16.37. ¹³C NMR (126MHz, CDCl₃) δ 167.9, 161.4, 155.1, 132.4, 124.6, 112.1, 63.9 (m), 44.7(t, J=146.8 Hz), 16.5 (m), 14.4. MS [EST⁺] m/z: 484.19 [M+H]⁺.

(((2-(3-((3-fluoro-4-methoxyphenyl)carbamoyl)phenyl)thieno[3,2-d]pyrimidin-4-yl)amino)methylene)bis(phosphonicacid) (I42) (Table 2)

Tetraethyl(((2-(3-((3-fluoro-4-methoxyphenyl)carbamoyl)phenyl)thieno[3,2-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate)was isolated as a light yellow solid (39.3 mg, 56%). ¹H NMR (500 MHz,CDCl₃) δ 9.37 (s, 1H), 9.26 (s, 1H), 8.56 (d, J=7.8 Hz, 1H), 8.09 (d,J=7.7 Hz, 1H), 7.71 (d, J=5.3 Hz, 1H), 7.67 (dd, J=13.1, 2.1 Hz, 1H),7.61 (d, J=8.5 Hz, 1H), 7.51 (t, J=7.7 Hz, 1H), 7.43 (d, J=5.3 Hz, 1H),6.86 (t, J=9.1 Hz, 1H), 6.54 (s, 1H), 5.62 (t, J=18.3 Hz, 1H), 4.15 (dd,J=42.1, 11.0 Hz, 8H), 3.83 (s, 3H), 1.18 (dd, J=13.0, 7.0 Hz, 12H). ³¹PNMR (203 MHz, CDCl₃) δ 17.06. ¹³C NMR (126 MHz, CDCl₃) δ 165.4, 161.4,159.7, 155.9 (t, J=3.78 Hz), 152.9, 150.9, 144.0 (d, J=11.34 Hz), 138.1,134.6, 132.5 (t, J=10.08 Hz), 130.9, 129.8, 128.8, 127.0, 125.3, 116.2(d, J=2.52 Hz), 114.2, 113.5 (d, J=2.52 Hz), 109.5 (d, J=22.68 Hz), 63.7(m), 56.6, 46.8 (t, J=148.7 Hz), 16.3 (m). MS (ESI⁺): (m/z) [M+H]⁺681.42; MS (ESI⁻): (m/z) [M−H]⁻ 679.30

After deprotection of the above tetraethyl ester intermediate, compoundI-42 was isolated as a white solid (15.8 mg, 57%).

¹H NMR (400 MHz, DMSO-d₆) δ 10.46 (s, 1H), 8.96 (s, 1H), 8.63 (d, J=7.8Hz, 1H), 8.24 (d, J=5.2 Hz, 1H), 8.05 (d, J=7.7 Hz, 1H), 7.79 (dd,J=13.8, 2.4 Hz, 1H), 7.69 (t, J=7.8 Hz, 1H), 7.55 (d, J=5.3 Hz, 2H),7.18 (t, J=9.4 Hz, 1H), 5.60-5.44 (m, 2H), 3.84 (s, 3H). ³¹P NMR (162MHz, DMSO-d₆) δ 13.46. HRMS (ESI−) calculated for C₂₃H₁₀FN₁₂P₂S m/z[M−H]⁻ 567.0337; found m/z 567.0328.

Ethyl 5-((ethoxycarbonyl)amino)thiazole-4-carboxylate (intermediate 18,Scheme 3); based on literature procedures (Shu, L. et al. Heterocycles.2012, 85, 1721-1726).

Anhydrous THF (10 ml) was added to a two-necked flask containing t-BuOK(546 mg, 4.86 mmol) under argon atmosphere. The mixture was cooled to−40° C. then ethyl isocyanoacetate (500 mg, 4.42 mmol) was addeddropwise at such that the temperature did not exceed −35° C. Afterwards,ethoxycarbonyl isothiocyanate (609 mg, 4.64 mmol) was also added to themixture dropwise. The resulting mixture was stirred for 1.5 hour lettingthe temperature free to rise to 0° C. The reaction was quenched viaaddition of glacial acetic acid (2.5 ml). The mixture was diluted withethyl acetate and water, extracted twice with ethyl acetate, washed withbrine and dried over sodium sulfate. The solution was concentrated undervacuo and purified by silica-gel column chromatography. The product wasobtained as an orange solid (821 mg, 76%). ¹H NMR (500 MHz, DMSO-d₆) δ10.01 (s, 1H), 8.59 (s, 1H), 4.33 (q, J=7.1 Hz, 2H), 4.26 (q, J=7.1 Hz,2H), 1.31 (t, J=7.1 Hz, 3H), 1.28 (t, J=7.1 Hz, 3H). ¹³C NMR (126 MHz,DMSO-d₆) δ 163.73, 152.59, 146.51, 145.11, 126.81, 62.73, 60.83, 14.18,14.13. MS [ESI⁺] m/z: 245.40 [M+H]⁺.

Ethyl (4-carbamoylthiazol-5-yl)carbamate (intermediate 19, Scheme 3);based on literature procedures (Shu, L. et al. Heterocycles. 2012, 85,1721-1726).

In a pressure vessel, compound 18 was dissolved in ethanol (0.8 ml) andstirred at 40° C. until all the solid was completely dissolved. To thissolution water (1.6 ml) was added, followed by ammonium hydroxide (5.8ml, 84 mmol), and then heated at 80° C. for 30 min. After cooling toroom temperature, the resulting solid was collected by filtration,rinsed with several portions of water, and dried in vacuo. Product 19was isolated as a white solid (510 mg, 71%). ¹H NMR (500 MHz, DMSO-d₆) δ10.89 (s, 1H), 8.58 (s, 1H), 7.83 (d, J=49.4 Hz, 2H), 4.23 (q, J=7.1 Hz,2H), 1.27 (t, J=7.1 Hz, 3H). MS [ESI⁺] m/z: 216.10 [M+H]⁺.

3a,7a-dihydrothiazolo[5,4-d]pyrimidine-5,7(4H,6H)-dione (intermediate20, Scheme 3); based on literature procedures (Shu, L. et al.Heterocycles. 2012, 85, 1721-1726).

A round-bottom flask was charged with t-BuOK (625.6 mg, 5.58 mmol) andN,N-dimethylacetamide (10 mL), followed by compound 19 (400 mg, 1.86mmol). The mixture was stirred at 100° C. under argon atmosphere for 1hour. The reaction was then cooled to room temperature and filtered. Theresidue was rinsed with water and dried in vacuo. The product wasisolated as an off-white solid (267 mg, 85%). ¹H NMR (500 MHz, DMSO-d₆)δ 11.97 (s, 1H), 11.29 (s, 1H), 8.71 (s, 1H). MS (ESI+): (m/z) [M+H]⁺170; MS (ESI⁻): (m/z) [M−H]⁻ 167.98

5,7-dichlorothiazolo[5,4-d]pyrimidine (intermediate 21, Scheme 3); basedon literature procedure by Arnott, E. A. et al. J. Org. Chem. 2011, 76,1653-1661.

A two-necked flask with a condenser was charged with compound 20 (500mg, 2.96 mmol) and purged with argon. Phosphorus oxychloride (4.1 mL,44.34 mmol) was then added at RT followed by the slow addition of DIPEA(0.7 ml, 3.84 mmol). The mixture was stirred at RT for 1 h and thenheated to 95° C. for 2.5 h. Phosphorus oxychloride was then distilledoff. The oily residue was then dissolved in ethyl acetate and washedwith aqueous sodium bicarbonate (2×). Organic layer were collected,dried with sodium sulfate and concentrated in vacuo. The product wasisolated as an off-white solid (353 mg, 58%). ¹H NMR (400 MHz, DMSO-d₆)δ 9.70 (s, 1H). MS [ESI⁺] m/z: 205.97 and 207.93 [M+H]⁺.

5-chloro-N-(2,4-dimethoxybenzyl)thiazolo[5,4-d]pyrimidin-7-amine(intermediate 22, Scheme 3)

A mixture of compound 21 (1.00 g, 4.85 mmol), dimethoxybenzylamine (1.06g, 6.31 mmol) and DIPEA (1.27 mL, 7.28 mmol) in DMSO (13 mL) was stirredat rt for 3 h. The mixture was poured into water (55 mL), cooled at 0°C. for 20 min and then filtered. The solid obtained was washed withwater. The product was then dried in vacuo. Crude product was purifiedby silica-gel column chromatography and isolated as a light yellowsolid. ¹H NMR (500 MHz, CDCl₃) δ 8.66 (s, 1H), 7.30 (d, J=8.1 Hz, 1H),6.81 (s, 1H), 6.44 (d, J=10.7 Hz, 2H), 4.73 (d, J=5.8 Hz, 2H), 3.84 (s,3H), 3.80 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 161.9, 160.9, 158.8,156.9, 156.3, 150.6, 131.0, 130.1, 117.7, 104.0, 98.7, 55.49, 55.46,40.5. MS [ESI⁺] m/z: 337.15 and 339.14 [M+H]⁺.

Synthesis for building blocks for the preparation of purine-based(Scheme 4) and pyrrolopyrimidine-based (Scheme 5) compounds, examplesinclude, but are not limited to examples I-44 and I-45, respectively, inTable 2

Synthesis of 2,6-dichloro-7H-purine (27, Scheme 4) was based onliterature procedures with minor modifications (for an example, refer toZheng, Q. et al. Org. Proc. Res. Dev., 2004, 8, 962-963.

Xanthine (26, 1.00 g, 6.58 mmol) and POCl₃ (6.20 mL, 66.8 mmol) weremixed at room temperature and then slowly heated to 50° C. under argon.To the reaction mixture, DBU (5.96 mL, 39.9 mmol) was added drop-wiseunder vigorous stirring. The mixture was heated to reflux (108° C.) for6 h (after 120 min all of xanthine was dissolved, and the reactionmixture formed a brown solution). Then the reaction mixture was cooledto 50° C. and slowly transferred to ice-water (70 g) under vigorousstirring. The brown solution obtained was neutralized to pH=4 with 50%aqueous NaOH solution and then filtered through a pad of Celite. Thelight yellow aqueous solution was extracted with ethyl acetate (2×40mL). The organic extracts were combined and concentrated under vacuum.Compound 27 (432 mg, 35% yield) was obtained as a yellow solid. ¹H-NMR(400 MHz, DMSO-d₆): δ 8.74 (s, 1H). MS (m/z): [M]⁺ 189.01

Synthesis of 2,6-dichloro-7-methyl-7H-purine (28) and2,6-dichloro-9-methyl-9H-purine (29, (Scheme 4)

To a solution of dichloropurine 27 (1.36 g, 7.20 mmol) was dissolved inacetone (22.7 mL) and potassium carbonate (1.49 mg, 10.8 mmol) was addedat room temperature. Methyl iodide (537 μl, 8.67 mmol) was added and themixture was stirred for 1.5 h at room temperature. The reaction mixturewas concentrated, water was added to the residue and stirred for 5 min,and then extracted with ethyl acetate (2×100 ml). The combined organiclayers were dried on MgSO₄, filtered and concentrated under reducedpressure. The crude residue was purified by column chromatography.Compound 29 was the major product formed (952 mg, 65%) and was obtainedas off-white solid and compound 28 was the minor product (389 mg, 27%)also isolated pure as an off-white solid; these results are consistantwith the literature (for example WO 2010/034706) Compound 29: ¹H-NMR(400 MHz, CDCl₃) δ 8.01 (s, 1H), 3.91 (s, 3H). MS (m/z): [M]⁺ 203.05.Compound 28: ¹H-NMR (400 MHz, CDCl₃) δ 8.20 (s, 1H), 4.16 (s, 3H). MS(m/z): [M]⁺ 203.05.

Synthesis of 2-chloro-N-(2,4-dimethoxybenzyl)-9-methyl-9H-purin-6-amine(intermediate 30, Scheme 4)

A solution of compound 29 (1.21 g, 5.96 mmol) in DMSO (15 mL) and2,4-dimethoxybenzylamine (1.16 mL, 7.74 mmol) was added DIPEA (1.78 mL,8.91 mmol) at room temperature and stirred at 6 h at the sametemperature. To the reaction mixture was added H₂O (20 mL) and shakenwell, a white precipitate was observed. Cooled the mixture at 0° C. for20 min filtered through the filter paper and the solid was washed withwater (3×). The precipitate was dried to obtain 30 (1.79 g, 90%) as awhite solid.

¹H-NMR (400 MHz, CD₃OD) δ 7.97 (s, 1H), 7.25 (d, J=8.2 Hz, 1H), 6.55 (d,J=2.3 Hz, 1H), 6.51-6.39 (m, 1H), 4.65 (s, 2H), 3.85 (s, 3H), 3.77 (s,3H), 3.75 (s, 3H); LCMS (m/z) [M+H]⁺333.24.

Synthesis of 1,7-dihydro-2H-pyrrolo[2,3-d]pyrimidine-2,4(3H)-dione(intermediate 33, Scheme 5) was based on literature procedures (Hatcher,J. M. et al. ACS Med. Chem. Lett. 2015, 6, 584-589); it would be obviousto chemists that the tautomer of 33 is7H-pyrrolo[2,3-d]pyrimidine-2,4-diol]

To a suspended solution of 6-aminouracil (32, 6.35 g, 50.0 mmol) andsodium acetate (4.10 g, 50.0 mmol) in H₂O (50 mL) at a temperature of72° C. was added a solution of chloroacetaldehyde (50% in water, 11.8 g,75.2 mmol) drop-wise. The reaction mixture was heated to 80° C. andstirring was continued for 60 min. After cooling the reaction mixture toroom temperature, the resulting solid was collected by filtration,washed with water and acetone, and dried under vacuum to afford 33 as alight-brown solid (6.46 g, 86%). ¹H-NMR (400 MHz, DMSO-d₆) δ 11.45 (s,1H), 11.10 (s, 1H), 10.48 (s, 1H), 6.64-6.51 (m, 1H), 6.22 (t, J=2.5 Hz,1H); MS (m/z) [M+H]⁺ 152.06.

Synthesis of 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (34, Scheme 5)

To a suspension of 33 (5.75 g, 38.0 mmol) in toluene (30 mL) underargon, was added POCl₃ (10.6 mL, 114 mmol). To the mixture DIPEA (13.3mL, 76.1 mmol) was added drop-wise over a period of 2.5 h at 70° C., andthen the temperature was increased to 108° C. and stirring continued for14 h. The reaction mixture was cooled to room temperature and thenpoured into a mixture of 150 mL ethyl acetate and 200 mL ice cold water,and then filtered through a pad of Celite. The aqueous layer wasextracted with ethyl acetate (3×200 mL) and the combined organic layerswere washed with brine and concentrated to give the 34 (3.42 g, 48%) asa light-brown solid. ¹H-NMR (400 MHz, DMSO-d₆) δ 12.79 (s, 1H),7.94-7.24 (m, 1H), 6.66 (ddd, J=5.3, 3.5, 1.7 Hz, 1H); MS (m/z): [M⁺]188.03.

Synthesis of 2,4-dichloro-7-methyl-7H-pyrrolo[2,3-d]pyrimidine (35,Scheme 5)

To a solution of 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine 34 (237 mg,1.26 mmol) in CH₃CN (1 mL) was added NaH (33.3 mg, 1.39 mmol)portion-wise at 0° C. The reaction mixture was stirred at roomtemperature for 20 min until gas evolution was ceased. Methyl iodide(86.4 μl, 1.39 mmol) was added and stirred the reaction mixture for 1 hat room temperature. To the reaction mixture was added water andextracted with ethyl acetate (2×30 mL). The combined organic layers weredried over MgSO₄, and then concentrated under vacuum. The resultantcrude residue was purified by silica-gel column chromatography to afford35 (144 mg, 58%) as white solid. ¹H-NMR (400 MHz, DMSO-d₆) δ 7.76 (d,J=3.6 Hz, 1H), 6.71 (d, J=3.6 Hz, 1H), 3.81 (s, 3H); MS: (m/z) [M+H]⁺204.02.

Synthesis of 2-chloro-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (36,Scheme 5)

To a solution of 35 (40.6 mg, 0.200 mmol) in dioxane (0.4 mL) in apressure vial was added 30% aqueous NH₄OH solution (1.5 mL). The mixturewas heated to 90° C. and stirred for 23 h. The reaction mixture wascooled to room temperature and then concentrated under vacuum. Theobtained crude residue was purified by silica-gel column chromatographyto afford 24 (32.8 mg, 89%) as a white solid. ¹H-NMR (400 MHz, DMSO-d₆)δ 7.45 (s, 2H), 7.11 (d, J=3.4 Hz, 1H), 6.52 (d, J=3.4 Hz, 1H), 3.64 (s,3H); MS (m/z): [M+H]⁺ 183.10.

Example 2: Expression and Purification of Recombinant hGGPPS

The hGGPPS enzyme was expressed and purified via a slightly modifiedprotocol described previously (Kavanagh, et al. J. Biol. Chem., 2006,281, 22004-22012). The plasmid containing N-terminallyhexahistidine-tagged human GGPPS was transformed into E. coli BL21 (DE3)competent cells containing kantamycin in Luria-Bertani (LB) medium andwas grown overnight at 37° C. Terrific broth medium (1L) containing 1 mLof 50 mM kantamycin was inoculated with 10 mL overnight seed culture andgrown at 37° C. until optical density (OD)₆₀₀ equals 1 at which pointthe temperature was reduced to 18° C. The culture was induced with 0.5mM isopropyl 1-thio-β-D-galactopyranoside and allowed to shakeovernight. Cells were pelleted via centrifugation and were incubated inthe freezer (−20° C.) overnight or at −80° C. until frozen. The frozencell pellet was subjected to the addition of protease inhibitor,Complete Mini-EDTA free pellet (Roche Life Science), and 20 mL ofbinding buffer (50 mM HEPES, 500 mM NaCl, 5 mM imidazole, 5% glycerol,10 mM β-mercaptoethanol, pH adjusted to 7.5). The cells were thensonicated, centrifuged and filtered. The His-tagged protein was loadedonto Ni-NTA agarose column, washed with binding buffer and eluted withbuffer containing (50 mM HEPES, 500 mM NaCl, 250 mM imidazole, 5%glycerol, 10 mM β-mercaptoethanol, pH adjusted to 7.5). The collectedprotein was further purified by gel filtration chromatography using aSuperdex 200 gel column with buffer containing 10 mM HEPES, 500 mM NaCl,5%-20% glycerol, 2 mM β-mercaptoethanol, pH adjusted to 7.5. The proteinwas concentrated with spin-column concentrator.

Example 3: In Vitro hGGPPS Inhibition Assay

The assay was based on a literature procedure (Kavanagh, et al. J. Biol.Chem., 2006, 281, 22004-22012) with minor modifications. All assays wererun in triplicate using recombinant human GGPPS (80 ng), FPP (10 μM),IPP (8.3 μM; ³H-IPP, 40 mCi/mmoL) in a final volume of 100 μL buffercontaining 50 mM Tris pH 7.7, 2 mM MgCl₂, 1 mM TCEP, 5 μg/mL BSA and0.2% (w/v) Tween 20. The enzyme and test compound were pre-incubated inthe assay buffer in a volume of 80 μL at 37° C. for 10 mins. Afterwards,the substrates (FPP, IPP) were added to start the reaction, which alsobring the compound, substrate, and buffer contents to the desired finalconcentrations as indicated above. The assay mixture was then incubatedat 37° C. for 15 mins (Note: the incubation time is based from the curvedetermined each time a new batch of enzyme is produced). Assays wereterminated by the addition of 200 μL of HCl/MeOH (1:4) and incubated for10 min at 37° C. The mixture was then extracted with 700 μL of petroleumether, dried through a plug of anhydrous Mg₂SO₄ and 300 μL of the driedligroin phase was combined with 8 mL of scintillation cocktail. Finally,the radioactivity was counted using a Beckman Coulter LS6500 liquidscintillation counter.

Reagents for the Enzymatic Assay: Petroleum ether (high boiling point,60°-80° C.) was purchased from Sigma Aldrich, liquid scintillationcocktail was purchased from MP Biomedicals (Ecolite Cat #: 882475),³H-IPP was obtained from American Radiolabeled Chemicals (ART 0377A; 1mCi/mL), and unlabeled IPP and FPP were purchased from Isoprenoids, Lc.as their tri-ammonium salts.

hGGPPS wild-type enzyme: The wild-type hGGPPS enzyme was stored at −80°C. as a 1 μg/μL stock solution in the eluent buffer (10 mM HEPES, pH7.5, 500 mM NaCl, 5%-20% glycerol, 2.0 mM β-mercaptoethanol).

IPP solution: ³H-IPP was diluted with unlabeled IPP (a.k.a. cold IPP) toa specific activity of 40 mCi/mmol and 82.7 μM concentration(radiolabeled+unlabeled IPP) in 10 mM Tris pH 7.7. It was stored at −10°C., warmed to 0° C. and kept on ice during the assay.

FPP solution: FPP was dissolved and diluted to a 100 μM concentration in10 mM Tris pH 7.7. It was stored at −10° C., warmed to 0° C. and kept onice during the assay.

In vitro hFPPS assay was carried out based on Method 2 (M2) aspreviously described (Leung et al. J. Med. Chem., 2013, 56, 7939-7950).Cell culture and viability assays and apoptosis in human myeloma celllines were also based on previous procedures (Leung et al. J. Med.Chem., 2013, 56, 7939-7950; Lin et al. J. Med. Chem., 2012, 55,3201-3215). Determination of total Tau and phosphorylated Tau levels inAD brain in the presence of test compounds at different concentrationsand LDH assays were also conducted based on previously describedprocedures (de Schutter et al. J. Med. Chem., 2014, 57, 5764-5776).

Results and Discussion

The results of the above biological screening assays are reported inTables 1, 2 and 3. The initial biological screening of representativecompounds of the application was carried out using a routine hGGPPSinhibition assay at a fixed concentration of 1 μM and 100 nM, inparallel with the literature compound 1 (ZOL) as the positive control.Representative examples are shown in Table 3. Selectivity against hFPPSand a full-dose IC₅₀ curves were determined for select compounds ofFormula I (Table 3). Consistent with previous observations (Leung et al.Bioorg. Med. Chem., 2013, 21, 2229-2240), substitution at C-2 or C-6(pyrimidine numbering) with a simple phenyl group (e.g. comparativecompounds C-5 and C-10) inhibit both hFPPS and hGGPPS enzymes withalmost equivalent potencies. A C-5 phenyl substituted analog, on theother hand, was inactive to both enzymes (data not shown). It shouldalso be noted that previous structure-activity relationship (SAR)studies focused on the identification of potent and selective inhibitorsof hFPPS, which suggested that substitution at C-5 with other aromaticgroups was unfavorable; consequently, substitution at C-5 was notexplored further. Replacement of the C-2 phenyl with heterocyclic groupsshowed that the more lipophilic moieties [phenyl comparative compoundC-5 and a corresponding C-2 thiophene comparative compound C-2 vs C-2pyridine comparative compound C-6) have better hGGPPS activity, ingeneral (Table 1)]. The C-2 and C-6 side chains were then expanded tooptimize the binding occupancy in consideration of the larger hGGPPSactive site and it was found that while the potency is almost the same,the bulkier substituents improved the selectivity to hGGPPS whenpositioned at C-2 (e.g. I-13 vs comparative compound C-5, Table 3). Onthe other hand, incorporation of the same amide substituent at the C-6position was found detrimental to the inhibitory activity for bothenzymes (e.g. comparative compound C-11 vs I-13, Table 3). Given theseresults, attention was focused on the C-2 position. Results for expandedanalogs that contain a sulfonamide- (e.g. I-11), urea (e.g. I-7) andamide-linker bearing a variety of different substituents (e.g.phenyl-based, for example, I-5, I-13, I-18, and, heterocycles I-3 andI-14, and cycloalkyl or heterocycloalkyl, for example I-4, I-15, I-30,I-46 and I-47) resulted in compounds with good activity for inhibitinghGGPPS (Table 2).

In summary, starting from the original “hits” (for example comparativecompound C-5, which is substituted with a phenyl at the C-2 of thethienopyrimidine core), selectivity was improved for inhibiting hGGPPSvs hFPPS by approximately 30-fold and 55-fold, in the case of compoundsI-13 and I-6, respectively (Table 3). Many of these compounds alsoinhibited the proliferation of MM human cancer cells with EC₅₀ values inthe submicromolar range (Table 3).

Example 4: Protocol for Multiple Myeloma (MM) Cell Culture and ViabilityAssays

Various MM cancer cell lines (Table 3 and FIGS. 5 and 6) were culturedin RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco BRL,Gaithersburg, Md.) supplemented with 2 mM L-glutamine in a 5% CO₂atmosphere at 37° C. EC₅₀ values for each target compound weredetermined using a commercial MTS proliferation assay (Promega, Madison,Wis.) following the manufacturer's instructions. Briefly, compounds werediluted directly into culture medium and then applied to cells that wereseeded in 96-well plates at a density 5,000 cells per well. Cells wereincubated with the indicated final concentration of compound in a totalmedia volume of 100 μl/well for 72 hours prior to the addition of theMTS reagent. Plates were incubated at 37° C. in the presence 5% CO₂ for2 hours prior to recording OD490 using a Tecan Infinite M200Promicroplate reader. Results were analyzed to obtain dose-response curvesand EC₅₀ calculations using GraphPad PRISM version 5 for the MacIntosh(GraphPad Software, San Diego, Calif.).

Example 5: Alternative Protocol for Various Cancer Cell Culture andViability Assays (Table 4)

1) Cell Culture

NCI-ADR-RES cells were obtained from National Cancer Institute (NCI) andall other cell lines were obtained from the American Type CultureCollection (ATCC). NHBE cells were maintained in Bronchial EpithelialCell Growth Medium (BEGM) supplemented with 10% FBS (Multicell) and 2 mML-glutamine (Gibco/Life Technologies). All other cells were maintainedin culture using Roswell Park Memorial Institute (RPMI) 1640 mediasupplemented with 10% FBS (Multicell), Penicillin (100 U/mL),Streptomycin (100 ug/mL) and 2 mM L-glutamine (Gibco/Life Technologies).Cells were plated in logarithmic growth phase in each well of clearbottom multiwell plates and cultured 16-24 h before drug treatment.Serial dilutions of compounds were made in DMSO before dilution inproper media and then added to cultured cells in order to reach amaximum of 0.2% DMSO. Cells were incubated with drugs for 72 hours.Information regarding the various cell lines tested is provided in theTable 4.

2) Determination of Cell Viability

Cell viability was determined by cellular quantitation of adenosinetriphosphate (ATP) using the CellTiter-Glo Luminescent Cell ViabilityAssay Kit (Promega Corporation, Cat. No. G7571). Briefly, theCellTiter-Glo «one-mix-measure» reagent, containing both the cellpermeabilizing agent and the Ultra-Glo luciferase, was added to the cellculture according to manufacturers' instructions allowing the free ATPto be released from viable cells and subsequently converted intoluminescence. The luminescence generated in the reaction is directlyproportional to cell viability and was quantified in a luminometer(PHERAstar FS, BMG Labtech)

3) Data Analysis

To calculate the relative growth inhibition induced by drug treatment,the mean value of relative light units (RLU) for replicate samples inthe CellTiter-Glo assay at each dose was divided by the mean RLU valueobtained from vehicle treated cells to give percent viability. Sigmoidaldose response curves and IC₅₀ values were generated using non-linearregression analysis (5 parameter fit) and GraphPad Prism Version 6(GraphPad Software Inc., San Diego, Calif.).

4) Results

The EC₅₀ values of compounds of the application are shown in FIGS. 4,and 5. FIG. 3, panels a and b indicate that compared to the commonantitumour drug doxorubicin, compounds of the application, exemplifiedby I-37 inhibit a variety of cancer types, are less toxic to normalhuman bronchial cells (NHBE; FIG. 3a ) and are equivalent or moreeffective in some cancer cell lines that are drug resistant todoxorubicin, such as ovarian cancer cells ADR-RES (FIG. 3b ).Additionally, FIG. 3c shows that compounds of the application,exemplified by I-37 inhibit a variety of cancer types. The data in FIG.4 shows that compound I-6 (panel a) is also more effective in blockingviability of cancer cells compared to zolendronate (ZOL) (panel b) forchronic myelogenous leukemia (K562) and acute monocytic leukemia(MOLM-13) cells. EC₅₀ values against MM cells RPMI-8226 for compoundsI-6, I-34, I-7, I-35, I-36, I-39, I-37, and I-40 are shown in FIG. 5.

Example 6: Apoptosis Assay In Multiple Myeloma Cell Assay

To determine the ability of compounds of the application to induce MMcell lines to undergo apoptosis, cells were seeded at a density of7.5×10⁵/mL in medium supplemented with 10% FBS with increasingconcentrations of compound (i.e. compound that inhibits hGGPPS) orvehicle alone. Following a 72 h incubation, apoptosis was determined bydouble staining with APC Annexin V (BD Biosciences, Mississauga ON) andeFluor 780 Viability dye (ThermoFisher Scientific) according to themanufacturer's directions. Stained samples were acquired on a BDFACSCanto II instrument (BD Biosciences, Mississauga ON) andpost-acquisition analyses were performed using FlowJo (V10) software.Apoptosis of multiple myeloma cells was also determined by flowcytometry by double staining cells with Allophycocyanin (APC) conjugatedAnnexin V and a V450 conjugated Mouse Anti-Human CD138 monoclonalantibody, following the manufacturer's instructions (BD Biosciences,Mississauga ON).

The apoptosis data of compound I-24 at different concentrations (100 nM,500 nM, and 1 μM) in multiple myeloma cells are shown in FIG. 6.Untreated cells and 5 nM Velcade were used as negative and positivecontrols, respectively. The data demonstrate that compounds of theapplication can induce apoptosis. However, the MM cells could be rescuedfrom apoptosis by the addition of geranylgeraniol (GGOH), but only whenapoptosis was induced by compounds of this application, not by velcade,which has a different mechanism of action. This observations (i.e. therescued of cells from apoptosis by the addition of GGOH as an externalsubstitute of the missing catalytic product of hGGPPS) confirmingspecificity of intracellular target engagement by the compounds disclosein this application.

Example 7: Western Blot Analysis

Multiple myeloma cells were cultured in RPMI-1640 media supplementedwith 10% FBS and L-glutamine and were maintained at 37° C. in 5% CO₂atmosphere in the presence and absence of the indicated concentration oftest compound. These experiments were performed to demonstrateintracellular down-regulation of Rap1A prenylation upon treatment withcompounds of this application. Geranylgeraniol co-treatment (GGOH; 10μM) served as a specificity control to demonstrate bypass of hGGPPSinhibitor treatment and specific target engagement in cells, which wasdirectly associated with hGGPPS inhibition. In other words, cells couldbe rescued from apoptosis upon co-treatment of these cells with a hGGPPSinhibitor plus GGOH. Cells were harvested by centrifugation after theindicated treatment duration. Harvested cells were immediately washedwith ice cold PBS, centrifuged, and then resuspended in ice cold RIPAlysis buffer (Pierce cat #89900). Equal amounts of cleared proteinlysate were then separated by SDS-PAGE, transferred to PVDF membranes,and then membranes were incubated with primary antibody overnight at 4°C. After extensive washing, membranes were exposed to HRP-conjugatedsecondary antibodies for 1 hour at room temperature. Finally, afterextensive washing standard chemiluminescence reagents and techniqueswere employed to visualize the bound secondary antibodies.

Example 8: Preclinical Profiling: Metabolic Stability in LiverMicrosomes of hGGPPS Inhibitors

As an example, the metabolic stability of inhibitor I-37 was evaluatedin liver microsomes from three species. After incubation of the compoundwith liver microsomes, the parent compound and any metabolites with abisphosphonate moiety were converted to the corresponding trimethylsilyl ester with trimethylsilyl diazomethane, following the protocolpreviously reported for the determination of bisphosphonate-type drugconcentrations in human plasma (see: Ghassabian, S. et al. J.Chromatogr. B 2012, 881-882, 34-41). Samples were analyzed by LC-MS/MSusing loperamide as a reference (half-life clearance of 8-15 min in allthree liver microsome incubations). The half-life clearance of I-37 inmale CD-1 mouse liver microsomes (MLM). Sprague-Dawley rat livermicrosomes (RLM) and human liver microsomes (HLM) was found to be 128min, 187 min and 154 min, respectively. Significantly higherlipophilicity was also observed for our thienopyrimidine hGPPS inhibitoras compare to current N-BP drugs. For example, a difference in relativeretention time of more than 7 min was observed on a C-18 reversed phaseHPLC column between compounds I-37 and the drug zolodronic acid,typically expected to translate into better cell-membrane permeabilityfor compound I-37 (by passive diffusion).

Example 9: In Vivo Study (Compound I-37)

In vivo experiments were performed according to the guidelines of theCanadian Council on Animal Care and approved by the Animal CareCommittee of the Research Institute of the McGill University HealthCentre as per protocol number 2012-7242. Mice were all bred andmaintained in a pathogen-free standard animal facility with a light/darkcycle of 12 hours and provided with food and water ad libitum.Vk*MYC/KaLwRij mice with an M-peak higher than 13% (and aged between 52and 77 weeks) were used for this in vivo study. Mice were placed ingroups of two (treated with 1 mg/kg, 5 mg/kg of I-37) and received a 17doses (15 day treatment course with a two day drug holiday during theweekend, followed by a two day re-treatment before sacrifice [number ofmice per group of 3, age and gender matched, intraperitoneal injection].Mice were euthanized after 24 h of receiving the last dose or asinstructed per protocol in case of toxicity effects. Mice wereanesthetized with ketamine and terminal blood collection was achieved bycardiac puncture. At cardiac puncture, whole blood from all mice wascollected in order to obtain serum and peripheral blood mononuclearcells (PBMCs).

Serum extraction: Blood was spun in a microcentrifuge at 6000 rpm for 15minutes to obtain serum. Serum was used to run Serum ProteinElectrophoresis (SPEP) and the remaining sample was stored in freezer at−80° C.

Isolation of peripheral blood mononuclear cells: Ammonium chloride wasused to lyse red blood cells (RBCs) from whole blood while PBMCs werenot thus lysed. Whole blood was diluted in 20 ml of ammonium chloride(155 mM NH₄Cl) and mixed by gentle vortexing for 10 minutes at roomtemperature. Cell solution was centrifuged at 400×g for 5 minutes andtwo washes with PBS were performed to remove ammonium chloride. PBMCswere harvested, lysed and analysed Western Blot to measure unprenylatedRap1A, following the same procedure as in the case of the MM RPMI-8226cells described above.

Example 10: Phosphorylation of the Tau Protein

As demonstrated in FIG. 7 and Table 5, compounds disclosed in thisapplication, exemplified by I-5, selectively inhibit hGGPPS anddownregulate phosphorylation of the tau protein (P-Tau) in humanimmortalized neurons (SH-SYSY neurons) to a greater extent thanequipotent inhibitors of hFPPS, such as compound 6-1 of WO2014/078957,in spite of structural similarities and very similar physicochemicalproperties such as Clog P values. Compared to zoledronic acid, thehGGPPS-selective I-5 is equally effective at reducing tau proteinphosphorylation but exhibits much lower toxicity (Table 5). Table 5contains inhibition data reflecting the decrease in phosphorylated Tauprotein to Total Tau protein levels in immortalized human neurons(SH-SYSY) treated with prior art compound 6-I (an hFPPS inhibitors ofWO2014/078957) compared to the exemplary hGGPPS inhibitor compound 6-I-5of the present application and their corresponding toxicity data tothese cells based on LDH toxicity. The experimental details for thesetypes of assays were previously reported by De Schutter et al. J. Med.Chem. 2014, 57, 5764-5776.

Different embodiments of the application have been shown by the aboveexample. Those skilled in the art could develop alternatives to themethods mentioned above that are within the scope of the application anddefined claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. Where a term in the present application is found to bedefined differently in a document incorporated herein by reference, thedefinition provided herein is to serve as the definition for the term.

TABLE 1 Structures and Activity Data for hGGPPS Inhibition forComparative and Reference Compounds hGGPPS Example STRUCTURE IC₅₀ (μM)Literature Compound 1 (mixture of E/Z isomers) Positive control

A Zolendronic acid (ZOL)

C Risedronic acid RIS

C C-1

B C-2

A C-3

C C-4

C C-5

A C-6

B C-7

B C-9

B C-10

A C-11

C C-12

B C-13

B A: IC₅₀ = ≤0.1 μM; B: 0.1-5 μM; C: IC₅₀ = ≥5 μM; ND = not determined

TABLE 2 Activity Data for hGGPPS Inhibition For Representative Compoundsof Formula I I-1

A I-2

B I-3

A I-4

B I-5

A I-6

A I-7

A I-8

A I-9

A I-10

B I-11

B I-12

B I-13

A I-14

B I-15

B I-16

B I-17

B I-18

A I-19

A I-20

B I-21

A I-22

A I-23

A I-24

A I-25

A I-26

B I-27

A I-28

A I-29

B I-30

A I-31

C I-32

B I-33

A I-34

A I-35

A I-36

A I-37

A I-38

B I-39

A I-40

B I-41

A I-42

A I-43

A I-44

B I-45

B I-46

A I-47

B I-48

B A: IC₅₀ = ≤0.1 μM; B: 0.1-5 μM; C: IC₅₀ = ≥5 μM; ND = not determined

TABLE 3 Potency and Selectivity of Select Compounds Ratio of PPS IC₅₀GGPPS IC₅₀ hFPPS IC₅₀ vs MM cells EC₅₀, μM^([b]) Compound (μM)(μM)^([a]) hGGPPS IC₅₀ JJN3 RPMI-8226 KMS28PE ZOL 0.004^([c])    >50 >1× 10⁻⁴ 5.0 11.0 6.4  RIS 0.011   ~350^([d]) >3 × 10⁻⁴ 10.0 13.0 10.6 C-5 0.54      0.082 ~7 nd nd nd C-10 0.20      0.094 ~2 nd nd ndC-11 >10    >10 nd nd >10 nd C-12 0.023 ~10,000 >4 × 10⁻⁵ >10 >50 ndC-13 0.014 ~10,000 >7 × 10⁻⁵ >10 >50 nd I-13 2.0      0.064 31 0.50 0.100.55 I-18 2.4      0.10 24 0.60 0.12 0.62 I-5 1.0      0.085 12 1.300.38 1.4  I-6 3.0      0.053 57 0.34 0.11 0.70 I-36 2.6      0.10 260.70 0.71 nd I-37 1.4      0.086 16 0.51 0.15 0.17 I-42 nd      0.085 —nd 0.20 nd I-43 nd      0.065 — nd 0.14 nd ^([a])IC₅₀ values weredetermined using the wild-type hGGPPS enzyme, average of n ≥ 3determinations; ^([b])Average of n ≥ 8 determinations, R² values in therange of 0.94-0.99; ^([c])IC₅₀ value from Kavanagh et. al. Proc. Natl.Acad. Sci. 2006, 103, 7829-7834; ^([d])IC₅₀ value from Szabo et. al. J.Med. Chem. 2002, 45, 2185-2196. nd = not determined

TABLE 4 Cell Lines Used in Viability Assays Cell Lines Cancer or NormalKRAS Status HT1080 Fibrosarcoma Activated (NRAS) HT-29 Colorectal WtKRAS overexpression MiaPaCa-2 Pancreatic Mutated HCT-116 ColorectalMutated T98G Brain MDA-MB-231 Breast Mutated A549 Non small cell lungMutated ADR-RES Ovarian Express high levels of multiple drug resistantpumps PC-3 Prostate NHBE Human bronchial cells (normal) RPMI-8226Multiple myeloma

TABLE 5 LDH Toxicity Assay Reduction in (relative toxicity comparedCompounds P-Tau/Total-tau protein to the control samples) Zoledronicacid 50% Very high  9% None observed 43% None observed

The invention claimed is:
 1. A compound of Formula I, or apharmaceutically acceptable salt, solvate and/or prodrug thereof:

wherein: R is selected from H, C₁₋₂alkyl and C₁₋₂fluoroalkyl; R¹ is(CR⁵R^(5′))PO(OR⁶)₂, wherein R⁵ is selected from PO(OR^(6′))₂,CO₂R^(6′), C(O)NHR⁷, SO₃R⁷ and SO₂NHR⁷; R^(5′) is selected from H, OHand halo; R⁶ and R^(6′) are independently selected from H and C₁₋₆alkyl;and R⁷ is selected from H, OH and C₁₋₆alkyl; X is selected from O, CH₂,NH and N(C₁₋₄alkyl); Z and Y are independently selected from S, O, NR³and CR³R^(3′); Cy¹ is selected from C₆₋₁₀aryl, C₅₋₁₀heteroaryl,C₃₋₁₀cycloalkyl and C₃₋₁₀heterocycloalkyl, each of which areunsubstituted or substituted with one or two substituents independentlyselected from halo, cyano, hydroxyl, NH₂, NHC₁₋₆alkyl, NHC₃₋₆cycloalkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), C₁₋₆fluoroalkyl, C₁₋₆alkyl, C₃₋₆cycloalkyl,C₁₋₆fluoroalkoxy, C₃₋₆cycloalkoxy and C₁₋₆alkoxy; Cy² is selected fromC₃₋₁₀cycloalkyl, C₃₋₁₀heterocycloalkyl, C₆₋₁₀aryl and C₅₋₁₀heteroaryl,each of which is unsubstituted or substituted with one to threesubstituents independently selected from halo, cyano, hydroxyl, NH₂,NHC₁₋₆alkyl, NHC₃₋₆cycloalkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), C₁₋₆fluoroalkyl,C₁₋₆alkyl, C₃₋₆cycloalkyl, C₁₋₆fluoroalkoxy, C₃₋₆cycloalkoxy, phenyl,C₃₋₆heterocycloalkyl, C₅₋₆heteroaryl and C₁₋₆alkoxy; L is selected froma direct bond, C(O), O, AC(O)(CR⁴R^(4′))_(m)(A′)_(p),ASO₂(CR⁴R^(4′))_(m)(A′)_(p), C(O)A(CR⁴R^(4′))_(m)(A′)_(p) andSO₂A(CR⁴R^(4′))_(m)(A′)_(p); R³ and R^(3′) are independently selectedfrom H, C₃₋₆cycloalkyl and C₁₋₄alkyl, or when the atom to which R³ isattached is sp₂ hybridized, R³ is not present; R⁴ and R^(4′) areindependently selected from H, halo, C₁₋₄fluoroalkyl, C₁₋₄alkyl,C₃₋₆cycloalkyl, C₁₋₄fluoroalkoxy, C₃₋₆cycloalkoxy and C₁₋₄alkoxy; m isselected from 0, 1 and 2; p is selected from 0 and 1; A is selected fromNH and N(C₁₋₄ alkyl); A′ is selected from O, NH and N(C₁₋₄ alkyl) when mis 1 or 2 and A′ is selected from NH and N(C₁₋₄ alkyl) when m is 0; and

represents a single or double bond, provided that two double bonds arenot adjacent to each other.
 2. The compound of claim 1, wherein R is H.3. The compound of claim 1, wherein R¹ is (CR⁵R^(5′))PO(OR⁶)₂, whereinR⁵ is selected from PO(OR^(6′))₂, CO₂R^(6′), C(O)NHR⁷, SO₃R⁷ andSO₂NHR⁷; R^(5′) is H; R⁶ and R^(6′) are independently selected from Hand CH₃; and R⁷ is selected from H, OH and CH₃.
 4. The compound of anyone of claim 1, having the following structure:


5. The compound of claim 1, wherein the bicyclic core is selected from:


6. The compound of claim 1, wherein, Cy¹ is selected from phenyl,thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl,isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl,1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl,1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl,1,3,4-thiadiazolyl, 1,3,4-oxadiazolyl, pyridinyl, pyrazinyl,pyrimidinyl, triazinyl, pyrrolidinyl, piperazinyl, piperidinyl,morpholinyl and pyridazinyl, each of which is unsubstituted orsubstituted with one or two substituents.
 7. The compound of claim 1,wherein L is selected from a direct bond, C(O), O,AC(O)(CR⁴R^(4′))_(m)(A′)_(p), ASO₂(CR⁴R^(4′))_(m)(A′)_(p),C(O)A(CR⁴R^(4′))_(m)(A′)_(p) and SO₂A(CR⁴R^(4′))_(m)(A′)_(p).
 8. Thecompound of claim 1, wherein R⁴ and R^(4′) are independently selectedfrom H, F, Cl, CF₃, CH₃, CF₃O and CH₃O.
 9. The compound of claim 1,wherein at least one of R⁴ and R^(4′) is H.
 10. The compound of claim 1,wherein A′ is O or NH when m is 1 or 2 and A′ is selected from NH andNCH₃ when m is
 0. 11. The compound of claim 1, wherein Cy² is selectedfrom phenyl, naphthyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,5-membered heteroaryl, 6-membered heteroaryl, 10-membered heteroaryl,5-membered heterocycloalkyl and 6-membered heterocycloalkyl.
 12. Thecompound of claim 1, wherein Cy¹ is unsubstituted or substituted onesubstituent selected from Cl, F, NHCH₃, N(CH₃)₂, CF₃, CH₃, CH₃CH₂,(CH₃)₂CH₂, CH₃O, CH₃CH₂O, (CH₃)₂CH₂O, CF₃O and CF₃O.
 13. The compoundclaim 1, wherein Cy² is unsubstituted or substituted with 1-3substituents independently selected from Cl, F, phenyl, cyano, hydroxyl,NH₂, NHCH₃, N(CH₃)₂, C₁₋₄fluoroalkyl, C₁₋₄alkyl, C₁₋₄fluoroalkoxy andC₁₋₄alkoxy.
 14. The compound of claim 1 selected from:

or pharmaceutically acceptable salts, solvates and prodrugs thereof. 15.A pharmaceutical composition comprising one or more compounds claim 1and a pharmaceutically acceptable carrier.
 16. A method of treating adisease, disorder or condition mediated by hGGPPS, or treatable byinhibiting geranylgeranylation of proteins, comprising administering atherapeutically effective amount of one or more compounds claim 1 to asubject in need thereof, wherein the disease, disorder or conditionmediated by hGGPPS, or treatable by inhibiting geranylgeranylation, iscancer, Alzheimer's Disease or osteoporosis.
 17. The method claim 16,wherein the disease, disorder or condition mediated by hGGPPS, ortreatable by inhibiting geranylgeranylation, is cancer.
 18. The methodof claim 17, wherein the cancer is a hematological cancer or a solidtumor cancer.
 19. The method of claim 17, wherein the cancer is multiplemyeloma, chronic myelogenous leukemia, acute monocytic leukemia, ovariancancer, pancreatic cancer, fibrosarcoma, colorectal cancer, brain canceror non-small cell lung cancer.